Indoleamine 2,3-dioxygenase based immunotherapy

ABSTRACT

The present invention relates to the field of prophylaxis and therapy of cancer. In particular there is provided a protein Indoleamine 2,3-dioxygenase (IDO) or peptide fragments here of that are capable of eliciting anti-cancer immune responses. Specifically, the invention relates to the use of IDO or peptides derived here from or IDO specific T-cells for treatment of cancer. The invention thus relates to an anti-cancer vaccine which optionally may be used in combination with other immunotherapies and to IDO specific T-cells adoptively transferred or induced in vivo by vaccination as a treatment of cancer. It is an aspect of the invention that the medicaments herein provided may be used in combination with cancer chemotherapy treatment. A further aspect relates to the prophylaxis and therapy of infections by the same means as described above. 
     The use of IDO and immunogenic peptide fragments hereof in cancer and infection treatment, diagnosis and prognosis is also provided.

This application is a Continuation of U.S. Ser. No. 12/988,124, filed 8Jul. 2011, which is a National Stage Application of PCT/DK2009/000095,filed 17 Apr. 2009, which claims benefit of Serial No. PA 2008 00565,filed 17 Apr. 2008 in Denmark and which applications are incorporatedherein by reference. To the extent appropriate, a claim of priority ismade to each of the above disclosed applications.

All patent and non-patent references cited in the application, or in thepresent application, are also hereby incorporated by reference in theirentirety.

FIELD OF INVENTION

The present invention relates to the field of prophylaxis and therapy ofcancer. In particular there is provided a protein Indoleamine2,3-dioxygenase (IDO) or peptide fragments here of that are capable ofeliciting anti-cancer immune responses. Specifically, the inventionrelates to the use of IDO or peptides derived here from or IDO specificT-cells for treatment of cancer. The invention thus relates to ananti-cancer vaccine which optionally may be used in combination withother immunotherapies and to IDO specific T-cells adoptively transferredor induced in vivo by vaccination as a treatment of cancer. It is anaspect of the invention that the medicaments herein provided may be usedin combination with cancer chemotherapy treatment. A further aspectrelates to the prophylaxis and therapy of infections by the same meansas described above.

The use of IDO and immunogenic peptide fragments hereof in cancer andinfection treatment, diagnosis and prognosis is also provided.

BACKGROUND OF INVENTION

The immune system has the capacity to recognize and destroy neoplasticcells; nevertheless, despite the fact that neoplastic transformation isassociated with the expression of immunogenic antigens, the immunesystem often fails to respond effectively to these antigens. The immunesystem obviously becomes tolerant towards these antigens¹. When thishappens, the neoplastic cells proliferate uncontrollably leading to theformation of malignant cancers with poor prognosis for the affectedindividuals. The acquired state of tolerance must be overcome for cancerimmunotherapy to succeed.

Indoleamine 2,3-dioxygenase (IDO) is a major component in maintainingthe homeostasis of the immune system which, however, also contributes totumor-induced tolerance. The expression and activation of IDO creates atolerogenic milieu in the tumor and the tumor-draining lymph nodes (LN)either via direct suppression of T cells by degradation of the essentialamino acid tryptophan or via enhancement of local Treg-(activatedregulatory T cells) mediated immunosuppression. With respect to theformer, some of the biological effects of IDO are mediated through localdepletion of tryptophan, whereas others are mediated viaimmunomodulatory tryptophan metabolites^(3,4). Recently, anIDO-responsive signaling system in T cells has been identified,comprising the stress kinase GCN2 5. GCN2 responds to elevations inuncharged tRNA, as would occur if the T cell were deprived oftryptophan, and T cells lacking GCN2 are refractory to IDO-mediatedsuppression and anergy induction⁶.

IDO can be expressed within the tumor by tumor cells as well as tumorstromal cells, where it inhibits the effector phase of immune responses.In addition, IDO-expressing antigen-presenting cells (APCs) are presentin tumor-draining lymph nodes, where they are believed to create atolerogenic microenvironment. Indeed, IDO-expressing CD19⁺ plasmacytoiddendritic cells (DCs) isolated from tumor-draining lymph node mediateprofound immune suppression and T cell anergy in vivo^(7,8);plasmacytoid DC from normal lymph nodes and spleen do not express IDO.Very few cells constitutively express IDO in normal lymphoid tissueexcept in the gut. This implies that the DCs in tumor-draining lymphnodes, which constitutively express IDO, must receive a stimulus whichis related to the presence of the tumor. This stimulus is believed to bedelivered by activated regulatory T cells (Tregs) migrating from thetumor to the draining lymph node. Tregs have been shown to induce IDOvia cell-surface expression of CTLA-4⁹. The induction of IDO convertsthe tumor-draining lymph nodes from an immunizing into a tolerizingmilieu. Indeed, when IDO⁺ DCs are injected in vivo, they createsuppression and anergy in antigen-specific T cells in the lymph nodesdraining the injection site¹⁰. Beside its expression in immune competentcells, IDO is frequently expressed in the tumor microenvironment, eitherin the tumor cells itself or in different stromal cells. In thissetting, IDO is believed to inhibit the effector phase of the immuneresponse^(11,12). In clinic, it has repeatedly been observed, thatexpression of IDO in tumor cells is associated with an impairedprognosis^(13,14).

Thus, the expression of IDO and the concomitant IDO induced cancerimmunosuppression poses a problem in the treatment of cancer.

SUMMARY OF INVENTION

The problem of cancer immunosuppression is solved by the presentinvention which is based on the surprising finding by the inventors ofspontaneous cytotoxic immune responses against IDO expressing cells incancer patients. These findings open the way for novel therapeutic anddiagnostic approaches which may be generally applicable in the controlof cancer diseases.

The present invention targets the cancer disease by killing the IDOexpressing cancer cells directly and by killing the IDO expressing,anergy inducing APCs/DCs. This is done by enabling the T cells torecognize the IDO expressing cells. Likewise, when the clinicalcondition is an infection, T cells are enabled to kill IDO expressingAPCs/DCs. Thus, the expression of the immune suppressing enzyme IDO incancer cells and APCs is positive in conjunction with the application ofthe method of the present invention, which targets these IDO expressingcells. This approach, especially as it entails the killing of theAPCs/DCs, goes against the common opinion in the field, where IDOgenerally is attempted down regulated/inhibited in order to remove thetolerizing milieu around the APCs/DCs while preserving these cells,which are considered required in order to launch an effective immuneresponse.

Furthermore, the finding of spontaneous cytotoxic immune responsesagainst IDO expressing cells is particularly surprising since IDOexpressing cells antagonize the desired effects of otherimmunotherapeutic approaches. Therefore, a combination of IDO− andtumor-targeting immunotherapies is highly synergistic.

The present invention regards a vaccine composition comprisingIndoleamine 2,3-dioxygenase (IDO) of SEQ ID NO: (1, 13, 14, 15 and/or16) or a functional homologue thereof having at least 70% identity toSEQ ID NO: (1, 13, 14, 15 and/or 16) or an immunogenically activepeptide fragment comprising a consecutive sequence of said IDO or saidfunctional homologue thereof or a nucleic acid encoding said IDO or saidpeptide fragment; in combination with an adjuvant for use as amedicament.

The synergistic effect of a combination of immunotherapies based on theabove disclosed vaccine is provided for in the aspect of the inventionwhich regards a kit-of-parts comprising the vaccine composition and afurther immunostimulating composition.

The aspect of combining the vaccine of the present invention with othercancer treatments such as chemotherapeutic agents is also provided forherein.

The aspect of combining the vaccine of the present invention with othertreatments against infections such as immunotherapies and/or antibioticsis also provided for herein.

Another aspect of the invention regards a composition for ex vivo or insitu diagnosis of the presence in a cancer patient of T cells in PBL orin tumor tissue that is reactive with IDO, the composition comprisingIndoleamine 2,3-dioxygenase (IDO) of SEQ ID NO (1, 13, 14, 15 and/or 16)or a functional homologue thereof having at least 70% identity to SEQ IDNO (1, 13, 14, 15 and/or 16) or an immunogenically active peptidefragment comprising a consecutive sequence of said IDO or saidfunctional homologue thereof.

A further aspect regards a diagnostic kit for ex vivo or in situdiagnosis of the presence in a cancer patient of T cells in PBL or intumor tissue that is reactive with IDO, the kit comprising Indoleamine2,3-dioxygenase (IDO) of SEQ ID NO (1, 13, 14, 15 and/or 16) or afunctional homologue thereof having at least 70% identity to SEQ ID NO(1, 13, 14, 15 and/or 16) or an immunogenically active peptide fragmentcomprising a consecutive sequence of said IDO or said functionalhomologue thereof.

Also a complex of a peptide fragment comprising a consecutive sequenceof said IDO or said functional homologue thereof and a Class I HLA or aClass II HLA molecule or a fragment of such molecule is provided forherein.

A method of detecting in a cancer patient the presence of IDO reactiveT-cells, the method comprising contacting a tumor tissue or a bloodsample with a complex of a peptide fragment comprising a consecutivesequence of said IDO or said functional homologue thereof and a Class IHLA or a Class II HLA molecule or a fragment of such molecule anddetecting binding of the complex to the tissue or the blood cells is afurther aspect of the present invention.

Yet an aspect of the invention regards a molecule that is capable ofbinding specifically to a peptide fragment comprising a consecutivesequence of said IDO or said functional homologue thereof.

It follows that a method of treating a clinical condition such as acancer or infection by any of the means described above falls within thescope of the present invention; the means including administering to anindividual suffering from the clinical condition an effective amount ofthe vaccine composition as disclosed above; a molecule that is capableof binding specifically to a peptide fragment comprising a consecutivesequence of said IDO or said functional homologue thereof or akit-of-parts comprising the aforementioned vaccine or molecule togetherwith another immunostimulating composition and/or chemotherapeuticagent.

It is thus also an object of the present invention to use animmunogenically active peptide fragment comprising a consecutivesequence of said IDO or said functional homologue thereof or the vaccinecomposition of above in the manufacture of a medicament for thetreatment or prevention of a cancer disease.

A further object of the present invention is a method of monitoringimmunization, said method comprising the steps of: providing a bloodsample from an individual; providing IDO of SEQ ID NO: (1, 13, 14, 15and/or 16) or a functional homologue thereof having at least 70%identity to SEQ ID NO: (1, 13, 14, 15 and/or 16) or an immunogenicallyactive peptide fragment comprising a consecutive sequence of said IDO orsaid functional homologue thereof or a nucleic acid encoding said IDO orsaid peptide fragment; determining whether said blood sample comprisesantibodies or T-cells comprising T-cell receptors specifically bindingthe protein or peptide; and thereby determining whether an immuneresponse to said protein or peptide has been raised in said individual.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1a shows HLA-A2-restricted T cell responses against IDO as measuredby IFN-γ ELISPOT. PBL from 13 healthy individuals, 4 breast cancerpatients, 6 melanoma patients, and 10 renal cell carcinoma patients wereanalyzed. All individuals were HLA-A2 positive. T lymphocytes werestimulated once with peptide IDO2 (FLVSLLVEI) (SEQ ID NO: 3) beforebeing plated at 4×10⁵ cells per well in duplicates either without orwith the peptide. The average number of peptide-specific spots (aftersubtraction of spots without added peptide) was calculated for eachpatient using the ImmunoSpot Series 2.0 Analyzer (CTL Analyzers).

FIG. 1b shows HLA-A2-restricted T cell responses against IDO as measuredby IFN-γ ELISPOT after T lymphocytes were stimulated once with peptideIDO6 (VLSKGDGL) (SEQ ID NO: 7), otherwise as described in FIG. 1 a.

FIG. 1c shows HLA-A2-restricted T cell responses against IDO as measuredby IFN-γ ELISPOT after T lymphocytes were stimulated once with peptideIDO5 (ALLEIASCL) (SEQ ID NO: 6), otherwise as described in FIG. 1 a.

FIG. 1d shows the number of IDO5-specific cells in PBMC measured byIFN-γ ELISPOT in correlation to IDO expression in the PBMC measured byintracellular IDO stainings.

FIG. 1e shows an example of an ELISPOT response against IDO5 in PBMCfrom a breast cancer patient.

FIG. 2a shows the binding affinity of the peptide IDO5 (SEQ ID NO: 6) toHLA-A2 compared to the HLA-A2-restricted positive control peptide HIV-1pol₄₇₆₋₄₈₄ (ILKEPVHGV) (SEQ ID NO: 18) by an assembly assay. Notably,IDO5 bound HLA-A2 even better than the high-affinity control epitope.The high binding affinity of IDO5 to HLA-A2 enabled preparation ofstable HLA-A2/IDO5 tetramers, which were used to detect IDO-reactive CTLby flow cytometry as shown in FIG. 2 b.

FIG. 2b shows flow cytometry of IDO5-specific CD8 T cells in PBL from arenal cell carcinoma patient after in vitro stimulation, with an HIVtetramer-complex used as control. This analysis clearly confirmed thepresence of IDO5-reactive CD8 T cells in the blood of HLA-A2 positivecancer patients.

FIG. 2c shows tetramer analysis of IDO5-specific T cells ex vivo and invitro in selected patients. In the three patients with strongestresponses after in vitro stimulation, a respective reactivity was alsodetected ex vivo. Overall, PBL from 7 HLA-A2 positive healthyindividuals and 11 HLA-A2 positive patients were analyzed which revealedan average frequency of 0.03% IDO reactive cells of total CD8+ T cellsafter in vitro stimulation in cancer patients, compared to 0.001% inhealthy donors.

FIG. 2d shows tetramer analysis of IDO5-specific T cells by flowcytometry with ex vivo phenotype staining of IDO5 tetramer gated cells.The ex vivo stainings of IDO-reactive T cells showed that naturallyoccurring IDO5-specific T cells have a CD45RA-CD28+ central/effectormemory phenotype. As a comparison the sample were stained with isotypematched controls.

FIG. 2e shows tetramer analysis of IDO5-specific T cells by flowcytometry illustrating the presence of IDO5− specific T cells in IL-2treated TIL cultures from HLA-A2+ melanoma and head and neck cancerpatients by tetramer stainings. IDO5-specific T cells could readily bedetected among the TIL. Overall, 4 of the 5 analyzed patients haddetectable IDO5-specific T cells.

FIG. 2f shows tetramer analysis of IDO5-specific T cells by flowcytometry. To control the specificity of the HLA-A2/IDO5 tetramer westained an IDO5-specific T-cell clone. The HLA-A2/IDO5 tetramer didefficiently stain the IDO5-specific T-cell clone, whereas the T-cellclone was not stained by the control HLAA2/HIV tetramer.

FIG. 3a shows specificity and functional capacity of an IDO5-specificT-cell clone (RBS35) assayed by ⁵¹Cr-release assay to detect lysis ofT2-cells with no peptide or pulsed with IDO5 peptide. The T-cell cloneRBS35 effectively killed IDO5-pulsed T2-cells whereas T2-cells withoutpeptide were not lysed, as described in example 3.

FIG. 3b shows specific lysis by IDO5-specific T-cell clone RBS35 whichkilled HLA-A2+, IDO+ colon cancer cell line SW480 with high efficacy. Incontrast, RBS35 did not lyse the HLA-A2+/IDO− colon cancer cell lineHCT-116, as described in example 4. HLA-restriction of RBS35 wasconfirmed by blocking HLA-class I using the HLA specific mAb W6/32,which completely abolished lysis of the SW480 target cells.

FIG. 3c shows specificity and functional capacity of an IDO5-specificT-cell clone (RBS35) assayed by ⁵¹Cr-release assay. Lysis of the IDO+,HLA-A2+ melanoma cell line FM55M without and with the addition of coldT2-cells pulsed with IDO5 or unpulsed (inhibitor to target ratio=20:1 isshown. The HLA-A2+/IDO+ melanoma cell line FM55M was killed by RBS35.Cold targeted inhibition assays using unlabeled T2-cells pulsed with theIDO5 (10 μM) peptide confirmed the HLA-A2/peptide-specificity of thekilling: The addition of cold (unlabeled) IDO5-pulsed T2-cellscompletely abrogated the killing of FM55M melanoma cells, whereas theaddition of cold T2-cells without peptide did not have an effect on thekilling of FM55M.

FIG. 3d shows specificity and functional capacity of an IDO5-specificT-cell clone (RBS35) assayed by ⁵¹Cr-release assay. The ability of RBS35 to lyse human HLA-A2+ AML-blasts enriched directly ex vivo from thebone-marrow of AML patients was tested. T cells (CD3+) and B cells(CD19+) were depleted from the bone marrow of HLA-A2+ AML patients usingCD19⁺ and CD3⁺ microbeads, respectively. The highly enriched AML-blasts(CD3−, CD19−) were subsequently used as target cells in a ⁵¹Cr releaseassay with or without the addition of the HLA-class I specific antibodyW6/32. RBS35 efficiently lysed the leukemia cells in an HLA-dependentmanner.

FIG. 3e shows histograms showing intracellular IDO expression in twocolon cancer cell lines: HCT116 and SW480. Intracellular IDO expressionwas given by a one-tailed two sampled T-test comparing MFIIDO (darkhistograms) and MFIIsotype control (light histograms), where MFI is theMean Fluorescence Intensity. Left: HCT-116 (p=0.300). Right: SW480(p=0.002).

FIG. 4a shows histograms of intracellular IDO stainings (darkhistograms) in colon cancer cell lines HCT-116 (0,01), and SW480 (1,3).Negative controls were stainings with the secondary fluorochromeconjugated antibody alone (light histograms). The IDO expression wasdetermined using the staining index defined asMFI_(positive)−MFI_(background)/2×SD_(background) where MFI is meanfluorescence intensity. Cells were defined IDO positive if the stainingindex>1.

FIG. 4b shows histograms of intracellular IDO stainings (darkhistograms) in breast cancer cell lines CAMA-1 (1,3), and CAMA-1+IFN-γ(1,8). Negative controls were stainings with the secondary fluorochromeconjugated antibody alone (light histograms). Cells were defined IDOpositive if the staining index>1.

FIG. 4c shows histograms of intracellular IDO stainings (darkhistograms) in immature dendritic cells (DC) (0,2), and mature dendriticcells (DC)(1,2). Negative controls were stainings with the secondaryfluorochrome conjugated antibody alone (light histograms). Cells weredefined IDO positive if the staining index>1.

FIG. 5a shows functional capacity of an IDO5-specific T-cell clone(RBS35) to kill an IFN-γ treated breast cancer cell line[s] assayed by⁵¹Cr-release assay. Lysis of the HLA-A2 positive breast cancer cell lineCAMA-1 before and after IFN-γ treatment is shown. The CAMA-1 cell linewas killed by RBS35. However, INF-γ treatment increased the expressionof IDO in the cell line. Thus INF-γ treatment increased the killing byRBS35 of CAMA-1.

FIG. 5b shows functional capacity of an IDO5-specific T-cell clone(RBS35) to kill an IFN-γ treated breast cancer cell line assayed by⁵¹Cr-release assay. Lysis of the HLA-A2 positive breast cancer cell lineMDA-MB-231 before and after IFN-γ treatment is shown. MDA-MB-231 was notrecognized by RBS35. However, INF-γ treatment increased the expressionof IDO, and INF-γ treatment introduced killing of the MDA-MB-231 cells.

FIG. 5c shows histograms showing intracellular IDO expression and HLA-A2expression in breast cancer cell line CAMA-1 before and after IFN-γtreatment.

FIG. 5d shows lysis of the colon cancer cell line SW480 transfected withIDO ShRNA for down-regulation of IDO protein expression by anIDO5-specific T-cell bulk culture. As a positive control, SW480 cellstransfected with control ShRNA were used as target cells. Cancer cellstransfected with IDO ShRNA were not recognized by the polyclonalIDO-specific bulk culture, whereas cells transfected with irrelevantcontrol ShRNA were killed.

FIG. 5e shows histograms showing intracellular IDO expression in SW480transfected with control ShRNA (p=0.001 and MFIIDO/MFIIsotypecontrol=4.8) (top) and IDO ShRNA (p=0.040 and MFIIDO/MFIIsotypecontrol=2.1) (bottom).

FIG. 6a shows functional capacity of an IDO5-specific T-cell clone(RBS35) to kill dendritic cells (DCs) assayed by ⁵¹Cr-release assay.RBS35 effectively killed the matured DC. In contrast, autologousimmature IDO− DC were not killed by RBS35.

FIG. 6b shows functional capacity of an IDO5-specific T-cell clone(RBS35) to kill dendritic cells (DCs) assayed by ⁵¹Cr-release assay.Lysis of HLA-A2+ allogeneic immature and mature DC is shown. Theallogenic matured DC were killed by RBS35 whereas the IDO− immature DCfrom the same donor were not affected.

FIG. 6c shows histograms showing intracellular IDO expression inimmature and in vitro matured DC. Mature DC exhibited expression of IDOin contrast to immature DC.

FIG. 6d shows the ability of RBS35 to lyse autologous monoctyes, T cellsand B cells. For this purpose, we isolated CD14+ monoctyes, CD3+ T cellsand CD19+ B cells directly ex vivo from IDO+ PBMC. The isolated cellswere subsequently used as target cells in a 51Cr-release assay withRBS35. As a control, in vitro generated autologous IDO− immatured DC andIDO+ matured DC were employed. Autologous CD14+ monoctyes, CD3+ T cellsand CD19+ B cells were not lysed by RBS35.

FIG. 6e shows HLA-A2 restricted T-cell responses against EBVBMLF1280-288 (GLCTLVAML) (SEQ ID NO: 19) as measured by ELISPOT in PBMCfrom a breast cancer patient. Cultures of PBMC were treated with IFN-γto increase the immune activity as well as IDO expression in thecultures with and without autologous IDO specific T cells. Five dayslater we examined the immune reactivity against the HLA-A2 restrictedimmunodominant epitope from EBV BMLF1280-288 (GLCTLVAML) (SEQ ID NO: 19)in the cultures. Although the overall cell number was the same in thecultures the reactivity against the EBV peptide was higher in thecultures with IDO-specific T cells.

FIG. 7a shows specificity and functional capacity of IDO5-specific Tcells assayed by 51Cr-release assays. Lysis by RBS35 of the HLA-A2+/IDO+melanoma cell line FM55M without and with the addition of cold T2-cellspulsed with IDO5 peptide or an irrelevant peptide (HIV-1 pol1476-484)(SEQ ID NO: 18) (inhibitor to target ratio=20:1), and NK cell activityof RBS35 examined using the natural killer cell line K562 as targetcells is shown. The addition of cold T2-cells pulsed with an irrelevantpeptide (HIV-1 pol476-484) (SEQ ID NO: 18) did not have an effect on thekilling of FM55M. No cytotoxicity was observed against the NK-celltarget cell line K562.

FIG. 7b shows lysis by RBS35 of AML-blasts enriched from six HLA-A2+ AMLpatients. AML-blasts, B cells, and T cells were depleted ex vivo fromthe bone marrow of the AML patients using CD19+ and CD3+ microbeads,respectively. The highly enriched AML-blasts were used as target cellswith or without the addition of the HLA-class I specific antibody W6/32.RBS35 efficiently lysed the HLA-A2+ leukemia cells in an HLA-dependentmanner, while HLA-A2− leukemia cells were not lysed

FIG. 7c shows lysis of T2-cells pulsed with IDO5 peptide or anirrelevant peptide (HIV-1 pol1476-484), and lysis of the HLAA2+/IDO+colon cancer cell line SW480 by an IDO5-specific T-cell bulk culture.

FIG. 7c shows lysis of T2-cells pulsed with IDO5 peptide or anirrelevant peptide (HIV-1 pol476-484), and lysis of the HLAA2+/IDO+colon cancer cell line SW480 by an IDO5-specific T-cell bulk culture. Toillustrate the representative killing of tumor targets by RBS35, thekilling of SW480 by a polyclonal, IDO5-specific bulk culture is shown.

FIG. 7d shows lysis of the HLA-A2+/IDO+ colon cancer cell line SW480 andHLA-A2+/IDO− colon cancer cell line HCT-116 by three differentIDO5-specific T-cell clones (RBS26 (white triangle), RBS31 (blacktriangle), RBS46 (grey triangle)) assayed by 51Cr-release assay. Allassays were performed in different E:T ratios. Similar to RBS35, none ofthese clones (RBS26, RBS31, RBS46) lysed the HLA-A2+/IDO− colon cancercell line HCT-116.

FIG. 8 shows alignment of IDO sequences IDO (SEQ ID NO: 1), IDOA (SEQ IDNO: 13), IDOB (SEQ ID NO: 14), and IDOC (SEQ ID NO: 15).

FIG. 9 shows pair wise alignment of IDO (SEQ ID NO: 1) and (SEQ ID NO:16).

DETAILED DESCRIPTION OF THE INVENTION

It is a major objective of the present invention to provide a vaccinecomposition comprising Indoleamine 2,3-dioxygenase (IDO) or animmunologically active polypeptide fragment hereof for use as amedicament in the prevention of, reduction of risk from, or treatment ofcancer.

DEFINITIONS

Adjuvant: Any substance whose admixture with an administered immunogenicdeterminant/antigen/nucleic acid construct increases or otherwisemodifies the immune response to said determinant.

Amino acid: Any synthetic or naturally occurring amino carboxylic acid,including any amino acid occurring in peptides and polypeptidesincluding proteins and enzymes synthesized in vivo thus includingmodifications of the amino acids. The term amino acid is herein usedsynonymously with the term “amino acid residue” which is meant toencompass amino acids as stated which have been reacted with at leastone other species, such as 2, for example 3, such as more than 3 otherspecies. The generic term amino acid comprises both natural andnon-natural amino acids any of which may be in the “D” or “L” isomericform.

Antibody: Immunoglobulin molecules and active portions of immunoglobulinmolecules. Antibodies are for example intact immunoglobulin molecules orfragments thereof retaining the immunologic activity.

Antigen: Any substance that can bind to a clonally distributed immunereceptor (T-cell or B-cell receptor). Usually a peptide, polypeptide ora multimeric polypeptide. Antigens are preferably capable of elicitingan immune response.

APC: Antigen-presenting cell. An APC is a cell that displays foreignantigen complexed with MHC on its surface. T-cells may recognize thiscomplex using their T-cell receptor (TCR). APCs fall into twocategories: professional, (of which there are three types: Dendriticcells, macrophages and B-cells) or non-professional (does notconstitutively express the Major histocompatibility complex proteinsrequired for interaction with naive T cells; these are expressed onlyupon stimulation of the non-professional APC by certain cytokines suchas IFN-γ).

Boost: To boost by a booster shot or dose is to give an additional doseof an immunizing agent, such as a vaccine, given at a time after theinitial dose to sustain the immune response elicited by the previousdose of the same agent.

Cancer: Herein any preneoplastic or neoplastic disease, benign ormalignant, where “neoplastic” refers to an abnormal proliferation ofcells.

Carrier: Entity or compound to which antigens are coupled to aid in theinduction of an immune response.

Chimeric protein: A genetically engineered protein that is encoded by anucleotide sequence made by a splicing together of two or more completeor partial genes or a series of (non)random nucleic acids.

Clinical condition: A condition that requires medical attention, hereinespecially conditions associated with the expression of IDO. Examples ofsuch conditions include: cancers and infections.

Complement: A complex series of blood proteins whose action“complements” the work of antibodies. Complement destroys bacteria,produces inflammation, and regulates immune reactions.

CTL: Cytotoxic T lymphocyte. A sub group of T-cells expressing CD8 alongwith the T-cell receptor and therefore able to respond to antigenspresented by class I molecules.

Cytokine: Growth or differentiation modulator, used non-determinativeherein, and should not limit the interpretation of the present inventionand claims. In addition to the cytokines, adhesion or accessorymolecules, or any combination thereof, may be employed alone or incombination with the cytokines.

Delivery vehicle: An entity whereby a nucleotide sequence or polypeptideor both can be transported from at least one media to another.

DC: Dendritic cell. (DCs) are immune cells and form part of themammalian immune system. Their main function is to process antigenmaterial and present it on the surface to other cells of the immunesystem, thus functioning as antigen-presenting cells (APCs).

Fragment: is used to indicate a non-full length part of a nucleic acidor polypeptide. Thus, a fragment is itself also a nucleic acid orpolypeptide, respectively.

Functional homologue: A functional homologue may be any nucleicacid/protein/polypeptide that exhibits at least some sequence identitywith a wild type version/sequence of a given gene/geneproduct/protein/polypeptide and has retained at least one aspect of theoriginal sequences functionality. Herein a functional homologue of IDOhas the capability to induce an immune response to cells expressing IDO.

IDO: Indoleamine 2,3-dioxygenase. Identified in SEQ ID NOs: (1, 13, 14,15, and 16).

Individual: Generally any species or subspecies of bird, mammal, fish,amphibian, or reptile, preferably a mammal, most preferably a humanbeing.

Infection: Herein the term “infection” relates to any kind of clinicalcondition giving rise to an immune response and therefore includesinfections, chronic infections, autoimmune conditions and allergicinflammations.

Isolated: used in connection with nucleic acids, polypeptides, andantibodies disclosed herein ‘isolated’ refers to these having beenidentified and separated and/or recovered from a component of theirnatural, typically cellular, environment. Nucleic acids, polypeptides,and antibodies of the invention are preferably isolated, and vaccinesand other compositions of the invention preferably comprise isolatednucleic acids, polypeptides or isolated antibodies.

MHC: Major histocompatibility complex, two main subclasses of MHC, ClassI and Class II exist.

Nucleic acid: A chain or sequence of nucleotides that convey geneticinformation. In regards to the present invention the nucleic acid is adeoxyribonucleic acid (DNA).

Nucleic acid construct: A genetically engineered nucleic acid. Typicallycomprising several elements such as genes or fragments of same,promoters, enhancers, terminators, polyA tails, linkers, polylinkers,operative linkers, multiple cloning sites (MCS), markers, STOP codons,other regulatory elements, internal ribosomal entry sites (IRES) orothers.

Operative linker: A sequence of nucleotides or amino acid residues thatbind together two parts of a nucleic acid construct or (chimeric)polypeptide in a manner securing the biological processing of thenucleic acid or polypeptide.

Pathogen: a specific causative agent of disease, especially a biologicalagent such as a virus, bacteria, prion or parasite that can causedisease to its host, also referred to as an infectious agent.

PBL: Peripheral blood cells are the cellular components of blood,consisting of red blood cells, white blood cells, and platelets, whichare found within the circulating pool of blood and not sequesteredwithin the lymphatic system, spleen, liver, or bone marrow.

PBMC: A Peripheral Blood Mononuclear Cell (PBMC) is a blood cell havinga round nucleus, such as a lymphocyte or a monocyte. These blood cellsare a critical component in the immune system to fight infection andadapt to intruders. The lymphocyte population consists of T cells (CD4and CD8 positive ˜75%), B cells and NK cells (˜25% combined).

Peptide: Plurality of covalently linked amino acid residues defining asequence and linked by amide bonds. The term is used analogously witholigopeptide and polypeptide. The natural and/or non-natural amino acidsmay be linked by peptide bonds or by non-peptide bonds. The term peptidealso embraces post-translational modifications introduced by chemical orenzyme-catalyzed reactions, as are known in the art. The term can referto a variant or fragment of a polypeptide.

Pharmaceutical carriers: also termed excipients, or stabilizers arenon-toxic to the cell or individual being exposed thereto at the dosagesand concentrations employed. Often the physiologically acceptablecarrier is an aqueous pH buffered solution. Examples of physiologicallyacceptable carriers include buffers such as phosphate, citrate, andother organic acids; antioxidants including ascorbic acid; low molecularweight (less than about 10 residues) polypeptide; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™

Plurality: At least two.

Promoter: A binding site in a DNA chain at which RNA polymerase binds toinitiate transcription of messenger RNA by one or more nearby structuralgenes.

Signal peptide: A short sequence of amino acids that determine theeventual location of a protein in the cell, also referred to as sortingpeptide.

Surfactant: A surface active agent capable of reducing the surfacetension of a liquid in which it is dissolved. A surfactant is a compoundcontaining a polar group which is hydrophilic and a non polar groupwhich is hydrophobic and often composed of a fatty chain.

Treg: Regulatory T cells/T lymphocytes

Vaccine: A substance or composition capable of inducing an immuneresponse in an animal. Also referred to as an immunogenic composition inthe present text. An immune response being an immune response(humoral/antibody and/or cellular) inducing memory in an organism,resulting in the infectious agent, being met by a secondary rather thana primary response, thus reducing its impact on the host organism. Avaccine of the present invention may be given as or prophylactic and/ortherapeutic medicament. The composition may comprise one or more of thefollowing: antigen(s), nucleic acid constructs comprising one or moreantigens operatively linked to li, carriers, adjuvants andpharmaceutical carriers.

Variant: a ‘variant’ of a given reference nucleic acid or polypeptiderefers to a nucleic acid or polypeptide that displays a certain degreeof sequence homology/identity to said reference nucleic acid orpolypeptide but is not identical to said reference nucleic acid orpolypeptide.

Indoleamine 2,3-dioxygenase

Indoleamine 2,3-dioxygenase (IDO) is an enzyme catalyzes the conversionof L-tryptophan to N-formylkynurenine and is thus the first and ratelimiting enzyme of tryptophan catabolism through the Kynurenine pathway.The catabolism of tryptophan causes a depletion of tryptophan whichsuppresses T-cell responses and promotes immune tolerance in mammalianpregnancy, tumor resistance, chronic infection, autoimmunity andallergic inflammation. Therefore, not only cancer, but infections ingeneral and infections, especially chronic infections, autoimmunity andallergic inflammations in particular are all clinical conditions ofrelevance for the present invention.

IDO is present in humans in five forms: IDO, IDOA, IDOB, IDOC andIDOLIKE (also known in the literature as IDO2). IDO is a 403 amino acidresidue long polypeptide as disclosed in SEQ ID NO:1, and is thepreferred IDO in the present text together with IDOLIKE of SEQ ID NO:16). The other IDOs are also of relevance to the present invention,although little is known about them; they are herein identified as IDOA:SEQ ID NO: 13, IDOB: SEQ ID NO: 14, and IDOC: SEQ ID NO 15. IDOLIKE isnot as widely expressed as IDO but like its relative is also expressedin antigen-presenting dendritic cells where tryptophan catabolism drivesimmune tolerance. As IDO, IDOLIKE catabolizes tryptophan, triggersphosphorylation of the translation initiation factor elF2alpha, andmobilizes translation of LIP, an inhibitory isoform of the immuneregulatory transcription factor NF-IL6 (Popov & Schultze, 2008). Hereinthe term IDO generally refers to all of the abovementioned IDOs andtheir corresponding sequences.

IDO has been identified as a major immune regulatory molecule, which ispart of several negative feedback mechanism by which immune responsesare kept under control. In this manner, IDO also exert criticalimmunosuppressive function in cancer. In the study underlying thepresent invention, it was examined whether IDO itself may serve astarget for immune responses, which may be exploited for immune therapy,particularly for treatment of cancer. By following a ‘reverseimmunology’ approach, HLA-A2 peptides within the IDO protein wereidentified to which spontaneous T-cell reactivity were detected inpatients suffering from unrelated tumor types, i.e. melanoma, renal cellcarcinoma and breast cancer. These naturally occurring T-cells responseswere readily visualized by flow cytometry using HLA/peptide tetramersafter in vitro stimulation but also in direct ex vivo assays.Furthermore, it was unequivocally confirmed that IDO reactive T cellsare indeed peptide specific, cytotoxic effector cells. In other words:IDO-specific T-cells effectively lyse IDO+ cancer cell lines ofdifferent origin, such us melanoma, colon carcinoma, breast cancer aswell as directly ex vivo enriched AML blasts. The presence ofspontaneous CTL-responses against IDO-derived peptide epitopes in PBLfrom patients suffering from unrelated cancer types as well as thekilling of cancer cells of different origin by IDO-specific T-cellsunderline the immunotherapeutic potential of IDO. Even more distinctiveis the finding that IDO-specific CTL recognize and kill IDO+ mature DC;hence, IDO-specific T-cells are able to kill immune competent DC. Recentreports have demonstrated that IDO is upregulated in human DCs uponmaturation^(23,24). Moreover, IDO expression is also observed in DCintended for vaccination in cancer patients and the expression ismaintained in situ after s.c. (subcutaneous) injection²⁵. Expression ofIDO in DC-based therapeutic vaccines holds critical clinical relevancevia the attraction or induction of FoxP3(+) Tregs.

The dual roles for IDO—inhibition of both the initial and the effectorphases of immune responses—are not mutually exclusive; in fact it islikely that both operate in a given tumor. There is an additional roleby which IDO may be highly relevant to the outcome of immunotherapy ofcancer: As an inflammation-induced counter-regulatory mechanism.Counter-regulatory responses are important in the immune system as theyhelp to limit the intensity and extent of immune responses, whichotherwise could cause dangerous damage to the host. However, with regardto anti-cancer immunotherapy counter-regulatory responses antagonize theability to create an intense immune response against the tumor.Counter-regulation differs from tolerance in the sense thatcounter-regulation is a secondary event, elicited only in response toimmune activation. IDO is known to be induced by both type I and IIinterferons, which are likely to be found at sites of immune activationand inflammation^(26,27). Notably, it is here in demonstrated that thatthe susceptibility of tumor cells to killing by IDO-reactive T-cells isincreased by pre-incubation with IFN-γ. Likewise, systemic ligation ofthe co-stimulatory molecule 4-1BB (CD137) has been reported to induceIDO²⁸. By definition most anti-cancer immunotherapeutic strategiesirrespective of their molecular targets aim at the induction of animmunological activation and inflammation (for example, at the site of avaccine or within the tumor). Virtually, within the limits of acceptabletoxicity, as much immune activation as possible is the goal; hence,counter-regulation is not desired. In this regard, in both melanoma andrenal cell carcinoma patients treatment with CTLA-4 blockade, i.e.anti-CTLA-4 antibodies, an association between enterocolitis and tumorregression has been reported²⁹. Hence, autoimmune reactions clearlycorrelate with clinical efficacy^(30,31). CTLA-4 blockade is thought tomediate its antitumor and IRAE-inducing effects by reducing peripheraltolerance to self antigens and increased T-cell activation by inhibitingthe function of Tregs.

Since IDO expressing antagonize the desired effects of otherimmunotherapeutic approaches, targeting IDO-expressing cells e.g. byvaccination, adoptive T-cell transfer or immune stimulatory agents, allof which are aspects of the present invention, consequently are highlysynergistic in action with additional anti-cancer immunotherapy. In thepresent disclosure it is demonstrated that CTL defined IDO epitopes arebroadly applicable in therapeutic vaccinations and are therefore ofsubstantial immunotherapeutic value.

It is thus an aspect of the present invention to provide a vaccinecomposition comprising Indoleamine 2,3-dioxygenase (IDO) or animmunologically active polypeptide fragment hereof for use as amedicament for the treatment of a clinical condition. Said clinicalcondition may be cancer and it is a further aspect of the presentinvention to prevent, reduce the risk from, or treat cancer. Anotheraspect relates to the use of the vaccine composition of the presentinvention in combination with other medicaments such asimmunotherapeutic medicaments and/or chemotherapeutic agents. Yet anaspect relates to the use of a vaccine composition as herein disclosedfor the treatment of diseases of viral and/or microbial origin andfurther to the use of said vaccine in combination with other medicamentssuch as immunotherapeutic medicaments and/or antibiotics and/oranti-viral agents.

Functional Homologues Sequences

The wild-type human IDO i.e. the naturally occurring non-mutated versionof the protein is identified in SEQ ID NO: 1. The present inventioncovers vaccine compositions comprising IDO; immunologically activepeptide fragments of IDO; peptide fragments of IDO, wherein at the mosttwo amino acids have been substituted; and/or functional homologues ofIDO comprising a sequence identity of at least 70% to SEQ ID NO: 1. Theterm polypeptide fragment is used herein to define any non-full length(as compared to SEQ ID NO: 1) string of amino acid residues that aredirectly derived from, synthesized to be identical with, or synthesizedto have a sequence identity of at least 70% with IDO as identified inSEQ ID NO:1.

A functional homologue can be defined as a full length or fragment ofIDO that differs in sequence from the wild-type IDO, such as wild-typehuman IDO, but is still capable of inducing an immune response againstIDO expressing cells such as cancer cells and DCs. The IDO expressed inthese cells may be wild type or endogenously mutated (such as acongenital mutant or a mutation induced during cell division or other).A functional homologue may be a mutated version or an alternative splicevariant of the wild-type IDO. In another aspect, functional homologuesof IDO are defined as described herein below. A functional homologue maybe, but is not limited to, a recombinant version of full length orfragmented IDO with one or more mutations and/or one or more sequencedeletions and/or additions introduced ex vivo.

A functional homologue of IDO may be any protein/polypeptide thatexhibits at least some sequence identity with SEQ ID NO: 1 and has thecapability to induce an immune response to cells expressing IDO.

Accordingly, in a specific embodiment the immunogenically active peptidefragment of the invention consists of 50 amino acid residues, forexample at the most 45 amino acid residues, such as at the most 40 aminoacid residues, for example at the most 35 amino acid residues, such asat the most 30 amino acid residues, for example at the most 25 aminoacid residues, such as 18 to 25 consecutive amino acids of IDO asidentified in SEQ ID NO: 1 or a functional homologue thereof; thefunctional homologue being one wherein at the most three amino acidshave been substituted, such as two amino acids, such as one amino acid.

Accordingly in another specific embodiment the immunogenically activepeptide fragment of the invention consists of the most 25 amino acidresidues, such as at the most 24 amino acid residues, such as at themost 23 amino acid residues, such as at the most 22 amino acid residues,such as at the most 21 amino acid residues, such as at the most 20 aminoacid residues, for example at the most 19 amino acid residues, such asat the most 18 amino acid residues, for example at the most 17 aminoacid residues, such as at the most 16 amino acid residues, for exampleat the most 15 amino acid residues, such as at the most 14 amino acidresidues, for example at the most 13 amino acid residues, such as at themost 12 amino acid residues, for example at the most 11 amino acidresidues, such as 8 to 10 consecutive amino acids from IDO of SEQ ID no1 or a functional homologue thereof; the functional homologue being onewherein at the most three amino acids have been substituted, such as twoamino acids, such as one amino acid. Preferably, the peptide comprisesat the most 10 consecutive amino acid residues from IDO, such as at themost 9 consecutive amino acid residues, such as 8 consecutive amino acidresidues, such as 7 consecutive amino acid residues from IDO asidentified in SEQ ID NO: 1 or a functional homologue thereof; thefunctional homologue being one wherein at the most three amino acidshave been substituted, such as two amino acids, such as one amino acid.

Accordingly in some embodiments the peptides of the invention arenonapeptides (peptides comprising 9 amino acid residues), and somedecapeptides (comprising 10 residues) and these may be selected from thenon-limiting group of (peptide name first, then sequence, place insequence of IDO and SEQ ID NO.): IDO1: Q L R E R V E K L (54-62) (SEQ IDNO: 2); IDO2: F L V S L L V E I (164-172) (SEQ ID NO: 3); IDO3: T L L KA L L E I (195-203) (SEQ ID NO: 4); IDO4: F I A K H L P D L (41-49) (SEQID NO: 5); IDO5: A L L E I A S C L (199-207) (SEQ ID NO:6); IDO6: V L SK G D A G L (320-328) (SEQ ID NO:7); IDO7: D L M N F L K T V (383-391)(SEQ ID NO: 8); IDO8: V L L G I Q Q T A (275-283) (SEQ ID NO: 9); IDO9:K V L P R N I A V (101-109) (SEQ ID NO: 10); IDO10: K L N M L S I D H L(61-70) (SEQ ID NO: 11); IDO11: S L R S Y H L Q I V (341-350) (SEQ IDNO: 12). Preferably, the peptide of the present invention is IDO5: A L LE I A S C L (199-207) (SEQ ID NO: 6); IDO2: F L V S L L V E I (164-172)(SEQ ID NO: 3); and/or IDO6: V L S K G D A G L (320-328) (SEQ ID NO: 7).Most preferably, vaccine composition of the present invention comprisesat least one peptide of IDO5: A L L E I A S C L (199-207) (SEQ ID NO:6).

Other peptides of the invention comprise (or more preferably consist of)between 4 and 120, preferably between 8 and 100, more preferably between10 and 75, yet more preferably between 12 and 60, even more preferablybetween 15 and 40, such as between 18 and 25 contiguous amino acids ofIDO of SEQ ID NO: 1 or a functional homologue thereof having at least70% identity to SEQ ID NO: 1, wherein at the most three amino acids ofIDO of SEQ ID NO: 1 have been substituted, deleted or added, such as twoamino acids have been substituted, deleted or added, or one amino acidhas been substituted, deleted or added.

In an embodiment of the invention IDO peptides comprise variantpeptides. As used herein the expression “variant” refers to peptideswhich are homologous to the basic protein, which is suitably human IDO,but which differs from the base sequence from which they are derived inthat one or more amino acids within the sequence are substituted forother amino acids. Suitably variants will have at the most six aminoacid substitutions, for example at the most five amino acidsubstitutions, such as at the most four amino acid substitutions, forexample at the most three amino acid substitutions, such as at the mosttwo amino acid substitutions, for example at the most one amino acidsubstitution.

Suitably variants will share at least 70% sequence identity to IDO ofSEQ ID NO: 1, and accordingly, variants preferably have at least 75%sequence identity, for example at least 80% sequence identity, such asat least 85% sequence identity, for example at least 90% sequenceidentity, such as at least 91% sequence identity, for example at least91% sequence identity, such as at least 92% sequence identity, forexample at least 93% sequence identity, such as at least 94% sequenceidentity, for example at least 95% sequence identity, such as at least96% sequence identity, for example at least 97% sequence identity, suchas at least 98% sequence identity, for example 99% sequence identitywith the sequence of human IDO.

Sequence identity can be calculated using a number of well-knownalgorithms and applying a number of different gap penalties. Thesequence identity is calculated relative to full-length SEQ ID NO: 1.Any sequence alignment tool, such as but not limited to FASTA, BLAST, orLALIGN may be used for searching homologues and calculating sequenceidentity. Moreover, when appropriate any commonly known substitutionmatrix, such as but not limited to PAM, BLOSSUM or PSSM matrices may beapplied with the search algorithm. For example, a PSSM (positionspecific scoring matrix) may be applied via the PSI-BLAST program.Moreover, sequence alignments may be performed using a range ofpenalties for gap opening and extension. For example, the BLASTalgorithm may be used with a gap opening penalty in the range 5-12, anda gap extension penalty in the range 1-2.

Functional equivalents may further comprise chemical modifications suchas ubiquitination, labeling (e.g., with radionuclides, various enzymes,etc.), pegylation (derivatization with polyethylene glycol), or byinsertion (or substitution by chemical synthesis) of amino acids (aminoacids) such as ornithine, which do not normally occur in human proteins,however it is preferred that the functional equivalent does not containchemical modifications.

Any changes made to the sequence of amino acid residues compared to thatof IDO of SEQ ID NO: 1 are preferably conservative substitutions. Aperson skilled in the art will know how to make and assess‘conservative’ amino acid substitutions, by which one amino acid issubstituted for another with one or more shared chemical and/or physicalcharacteristics. Conservative amino acid substitutions are less likelyto affect the functionality of the protein. Amino acids may be groupedaccording to shared characteristics. A conservative amino acidsubstitution is a substitution of one amino acid within a predeterminedgroup of amino acids for another amino acid within the same group,wherein the amino acids within a predetermined groups exhibit similar orsubstantially similar characteristics.

Thus, in an embodiment of the present invention, the vaccine compositioncomprises a polypeptide consisting of a consecutive sequence of IDO ofSEQ ID NO: 1 in the range of 8 to 50 amino acids, preferably in therange of 8 to 10 or 20 to 25 amino acids, wherein at the most threeamino acid has been substituted, and where the substitution preferablyis conservative.

MHC

There are two types of MHC molecules; MHC class I molecules and MHCclass II molecules. MHC class I molecules are recognized by CD8 T-cells,which are the principal effector cells of the adaptive immune response.MHC class II molecules are mainly expressed on the surface of antigenpresenting cells (APCs), the most important of which appears to be thedendritic cells. APCs stimulate naïve T-cells, as well as other cells inthe immune system. They stimulate both CD8 T-cells and CD4 T-cells.

In one embodiment, there are provided novel MHC Class I-restrictedpeptide fragments consisting of 8-10 amino acids from IDO of SEQ ID NO 1or a functional homologue thereof, wherein at the most two amino acidsof SEQ ID NO 1 have been substituted, which are characterized by havingat least one of several features, one of which is the ability to bind tothe Class I HLA molecule to which it is restricted at an affinity asmeasured by the amount of the peptide that is capable of half maximalrecovery of the Class I HLA molecule (C₅₀ value) which is at the most 50μM as determined by the assembly binding assay as described herein. Thisassembly assay is based on stabilization of the HLA molecule afterloading of peptide to the peptide transporter deficient cell line T2.Subsequently, correctly folded stable HLA heavy chains areimmunoprecipitated using conformation dependent antibodies and thepeptide binding is quantitated. The peptides of this embodimentcomprises (or more preferably consists of) at the most 200, preferablyat the most 100, more preferably at the most 50, yet more preferably atthe most 25, even more preferably at the most 20, yet even morepreferably at the most 15, such as at the most 10, for example in therange of 8 to 10 contiguous amino acids of IDO of SEQ ID NO 1 or afunctional homologue thereof wherein at the most two amino acids of SEQID NO 1 have been substituted.

This assay provides a simple means of screening candidate peptides fortheir ability to bind to a given HLA allele molecule at the aboveaffinity. In preferred embodiments, the peptide fragment of theinvention in one having a C₅₀ value, which is at the most 30 μM, such asa C₅₀ value, which is at the most 20 μM including C₅₀ values of at themost 10 μM, at the most 5 μM and at the most 2 μM.

In another preferred embodiment, there are provided novel MHC ClassII-restricted peptide fragments of IDO of SEQ ID NO 1 or a functionalhomologue thereof, wherein at the most two amino acids of SEQ ID NO 1have been substituted, (also referred to herein as “peptides”), whichare characterized by having at least one of several features describedherein below. The peptides of this embodiment comprises (or morepreferably consists of) between 4 and 120, preferably between 8 and 100,more preferably between 10 and 75, yet more preferably between 12 and60, even more preferably between 15 and 40, such as between 18 and 25contiguous amino acids of IDO of SEQ ID NO 1 of SEQ ID NO 1 or afunctional homologue thereof, wherein at the most two amino acids of SEQID NO 1 have been substituted,

Thus there are provided novel MHC Class I-restricted peptide fragmentsof 8-10 amino acids or novel MHC Class II-restricted peptide fragmentsof 18-25 amino acids of IDO of SEQ ID NO 1 or a functional homologuethereof, wherein at the most two amino acids of SEQ ID NO 1 have beensubstituted, which are characterized by having at least one of severalfeatures described herein below, one of which is the ability to bind tothe Class I or Class II HLA molecule to which it is restricted.

In particular embodiments there are provided peptide fragments, which isan MHC Class I-restricted peptide or an MHC class II-restricted peptidehaving at least one of the following characteristics:

-   -   (i) capable of eliciting INF-γ-producing cells in a PBL        population of a cancer patient at a frequency of at least 1 per        10⁴ PBLs as determined by an ELISPOT assay, and/or    -   (ii) capable of in situ detection in a tumor tissue of CTLs that        are reactive with the epitope peptide.    -   (iii) capable of inducing the growth of IDO specific T-cells in        vitro.

More preferred peptides according to the present invention are peptidescapable of raising a specific T-cell response as determined by anELISPOT assay, for example the ELISPOT assay described in Example 1herein below. Some peptides although they do not bind MHC class I orclass II with high affinity, may still give rise to a T-cell response asdetermined by ELISPOT. Other peptides capable of binding MHC class I orclass II with high affinity also give rise to a T-cell response asdetermined by ELISPOT. Both kinds of peptides are preferred peptidesaccording to the invention.

Hence, preferred peptides according to the present invention arepeptides capable of raising a specific T-cell response as measured by anELISPOT assay, wherein more than 50 peptide specific spots per 10⁸cells, more preferably per 10⁷, even more preferably per 10⁶, yet morepreferably per 10⁵ cells, such as per 10⁴ cells are measured.

Most preferred peptides according to the present invention are peptidesthat are capable of eliciting a cellular immune response in anindividual suffering from a clinical condition characterized by theexpression of IDO, the clinical condition preferably being a cancer orinfection, and most preferably a cancer.

As described above, the HLA system represents the human majorhistocompatibility (MHC) system. Generally, MHC systems control a rangeof characteristics: transplantation antigens, thymus dependent immuneresponses, certain complement factors and predisposition for certaindiseases. More specifically, the MHC codes for three different types ofmolecules, i.e. Class I, II and III molecules, which determine the moregeneral characteristics of the MHC. Of these molecules, the Class Imolecules are so-called HLA-A, HLA-B and HLA-C molecules that arepresented on the surface of most nucleated cells and thrombocytes.

The peptides of the present invention are characterized by their abilityto bind to (being restricted by) a particular MHC Class I HLA molecule.Thus, in one embodiment the peptide is one which is restricted by a MHCClass I HLA-A molecule including HLA-A1, HLA-A2, HLA-A3, HLA-A9,HLA-A10, HLA-A11, HLA-Aw19, HLA-A23(9), HLA-A24(9), HLA-A25(10),HLA-A26(10), HLA-A28, HLA-A29(w19), HLA-A30(w19), HLA-A31(w19),HLA-A32(w19), HLA-Aw33(w19), HLA-Aw34(10), HLA-Aw36, HLA-Aw43,HLA-Aw66(10), HLA-Aw68(28), HLA-A69(28). More simple designations arealso used throughout the literature, where only the primary numericdesignation is used, e.g. HLA-A19 or HLA-A24 instead of HLA-Aw19 andHLA-A24(49), respectively. In specific embodiments, the peptide of theinvention is restricted a MHC Class I HLA species selected from thegroup consisting of HLA-A1, HLA-A2, HLA-A3, HLA-A11 and HLA-A24. Inspecific embodiment, the peptide of the invention is restricted a MHCClass I HLA species HLA-A2 or HLA-A3.

In further useful embodiments, the peptide of the invention is apeptide, which is restricted by a MHC Class I HLA-B molecule includingany of the following: HLA-B5, HLA-B7, HLA-B8, HLA-B12, HLA-B13, HLA-B14,HLA-B15, HLA-B16, HLA-B17, HLA-B18, HLA-B21, HLA-Bw22, HLA-B27, HLA-B35,HLA-B37, HLA-B38, HLA-B39, HLA-B40, HLA-Bw41, HLA-Bw42, HLA-B44,HLA-B45, HLA-Bw46 and HLA-Bw47. In specific embodiments of theinvention, the MHC Class I HLA-B species to which the peptide of theinvention is capable of binding is selected from HLA-B7, HLA-B35,HLA-B44, HLA-B8, HLA-B15, HLA-B27 and HLA-B51.

In further useful embodiments, the peptide of the invention is apeptide, which is restricted by a MHC Class I HLA-C molecule includingbut not limited to any of the following: HLA-Cw1, HLA-Cw2, HLA-Cw3,HLA-Cw4, HLA-Cw5, HLA-Cw6, HLA-Cw7 and HLA-Cw1.

In further useful embodiments, the peptide of the invention is apeptide, which is restricted by a MHC Class II HLA molecule includingbut not limited to any of the following: HLA-DPA-1, HLA-DPB-1, HLA-DQA1,HLA-DQB1, HLA-DRA, HLA-DRB and all alleles in these groups and HLA-DM,HLA-DO.

The selection of peptides potentially having the ability to bind to aparticular HLA molecule can be made by the alignment of known sequencesthat bind to a given particular HLA molecule to thereby reveal thepredominance of a few related amino acids at particular positions in thepeptides. Such predominant amino acid residues are also referred toherein as “anchor residues” or “anchor residue motifs”. By followingsuch a relatively simple procedure based on known sequence data that canbe found in accessible databases, peptides can be derived from IDO,which are likely to bind to a specific HLA molecule. Representativeexamples of such analyses for a range of HLA molecules are given in thebelow table:

TABLE 2 Position Position Position Position Position Position C- HLAallele 1 2 3 5 6 7 terminal HLA-A1 T, S D, E L Y HLA-A2 L, M V L, VHLA-A3 L, V, M F, Y K, Y, F HLA-A11 V, I, F, Y M, L, F, Y, I K, RHLA-A23 I, Y W, I HLA-A24 Y I, V F I, L, F HLA-A25 M, A, T I W HLA-A26E, D V, T, I, L, F I, L, V Y, F HLA-A28 E, D V, A, L A, R HLA-A29 E Y, LHLA-A30 Y, L, F, V Y HLA-A31 L, M, F, Y R HLA-A32 I, L W HLA-A33 Y, I,L, V R HLA-A34 V, L R HLA-A66 E, D T, V R, K HLA-A68 E, D T, V R, KHLA-A69 V, T, A V, L HLA-A74 T V, L HLA-B5 A, P F, Y I, L HLA-B7 * P L,F HLA-B8 K K, R L HLA-B14 R, K L, V HLA-B15 Q, L, K, P, F, Y, W (B62) H,V, I, M, S, T HLA-B17 L, V HLA-B27 R Y, K, F, L HLA-B35 P I, L, M, YHLA-B37 D, E I, L, M HLA-B38 H D, E F, L HLA-B39 R, H L, F HLA-B40 E F,I, V L, V, A, W, (B60, 61) M, T, R HLA-B42 L, P Y, L HLA-B44 E F, Y, WHLA-B46 M, I, L, V Y, F HLA-B48 Q, K L HLA-B51 A, P, G F, Y, I, VHLA-B52 Q F, Y I, V HLA-B53 P W, F, L HLA-B54 P HLA-B55 P A, V HLA-B56 PA, V HLA-B57 A, T, S F, W, Y HLA-B58 A, T, S F, W, Y HLA-B67 P L HLA-B73R P HLA-Cw1 A, L L HLA-Cw2 A, L F, Y HLA-Cw3 A, L L, M HLA-Cw4 Y, P, FL, M, F, Y HLA-Cw6 L, I, V, Y HLA-Cw6 Y L, Y, F HLA-Cw8 Y L, I, HLA-Cw16A, L L, V * In one embodiment there is no specific anchor residue forthis position, however in a preferred embodiment the anchor residue is Ror A.

Thus, as an example, nonapeptides potentially having the ability to bindto HLA-A3 would have one of the following sequences:Xaa-L-Y-Xaa-Xaa-Xaa-Xaa-Xaa-K, Xaa-L-Y-Xaa-Xaa-Xaa-Xaa-Xaa-Y;Xaa-L-Y-Xaa-Xaa-Xaa-Xaa-Xaa-F or Xaa-V-Y-Xaa-Xaa-Xaa-Xaa-Xaa-K (Xaaindicating any amino acid residue). In a similar manner, sequencespotentially having the ability to bind to any other HLA molecule can bedesigned. It will be appreciated that the person of ordinary skill inthe art will be able to identify further “anchor residue motifs” for agiven HLA molecule.

The peptide of the invention may have a sequence which is a nativesequence of the IDO from which is derived. However, peptides having ahigher affinity to any given HLA molecule may be derived from such anative sequence by modifying the sequence by substituting, deleting oradding at least one amino acid residue, e.g. on the basis of theprocedure described above, whereby anchor residue motifs in respect ofthe given HLA molecule are identified.

Thus, in useful embodiments, the polypeptides of the invention includepeptides, the sequences of which comprise, for each of the specific HLAalleles listed in the table, any of the amino acid residues as indicatedin the table.

Thus, the peptides of the invention may be any of the above-mentionedpeptides comprising contiguous sequences from IDO, wherein in the rangeof 1 to 10, preferably in the range of 1 to 5, more preferably in therange of 1 to 3, even more preferably in the range of 1 to 2, yet morepreferably 1 amino acid has been exchanged for another amino acid,preferably in a manner so that the peptide comprises one or more,preferably all anchor residues of a given HLA-A specific peptide asindicated in the table above.

Examples preferable HLA species include, to which preferred peptides ofthe present invention are restricted include: a MHC Class I HLA speciesselected from the group consisting of HLA-A1, HLA-A2, HLA-A3, HLA-A11and HLA-A24, more preferably the peptide is restricted by HLA-A3 orHLA-A2. Alternatively a preferred HLA species includes MHC Class I HLA-Bspecies selected from the group consisting of HLA-B7, HLA-B35, HLA-B44,HLA-B8, HLA-B15, HLA-B27 and HLA-B51.

An approach to identifying polypeptides of the invention includes thefollowing steps: selecting a particular HLA molecule, e.g. one occurringat a high rate in a given population, carrying out an alignment analysisas described above to identify “anchor residue motifs” in the IDOprotein, isolating or constructing peptides of a suitable size thatcomprise one or more of the identified anchor residues and testing theresulting peptides for the capability of the peptides to elicitINF-γ-producing cells in a PBL population of a cancer patient at afrequency of at least 1 per 10⁴ PBLs as determined by an ELISPOT assayas described in Example 1.

In one aspect of the present invention, IDO-derived peptides longer than8 to 10 amino acid residues are provided. Polypeptides longer than 8 to10 amino acids are processed by the proteasome to a shorter length forbinding to HLA molecules. Thus, when administering a polypeptide longerthan 8 to 10 amino acid residues long, the “long”polypeptide/protein/protein fragment/variant of IDO is processed into aseries of smaller peptides in the cytosol by the proteasome. Anadvantage of using a longer polypeptide that may be processed by theproteasome into a variety of different shorter peptides is that more HLAclasses may be targeted with one peptide than one 8 to 10 amino acidpeptide that is restricted to a particular HLA class.

Surprisingly, some of the peptides of the present invention bind to MHCmolecules with an affinity sufficiently high to render substitutionsunnecessary (see FIG. 2) and are ready for use as antigens as they arepresented here. Preferably, the vaccine composition of the presentinvention comprises one or more of the following: IDO protein (SEQ IDNO: 1), polypeptide fragments here from, likewise variants, functionalhomologues of full length and partial length IDO, contiguous peptides ofIDO and functional homologues of these. More preferably, the vaccinecomposition comprises any of the sequences listed in the sequence listof the present disclosure. Very preferably, the vaccine compositioncomprises the peptides IDO5 (SEQ ID NO: 6), IDO 2 (SEQ ID NO: 3), and/orIDO6 (SEQ ID NO: 7).

A significant feature of the peptide of the invention is its capabilityto recognize or elicit INF-γ-producing responder T cells, i.e. cytotoxicT cells (CTLs) that specifically recognize the particular peptide in aPBL population, on an APC or tumor/neoplastic cells of an individualsuffering from a cancer and/or an infection (target cells). Thisactivity is readily determined by subjecting PBLs, APCs or tumor cellsfrom an individual to an ELISPOT assay. Prior to the assay, it may beadvantageous to stimulate the cells to be assayed by contacting thecells with the peptide to be tested. Preferably, the peptide is capableof eliciting or recognizing INF-γ-producing T cells at a frequency of atleast 1 per 10⁴ PBLs as determined by an ELISPOT assay as used herein.More preferably the frequency is at least 5 per 10⁴ PBLs, mostpreferably at least 10 per 10⁴ PBLs, such as at least 50 or 100 per 10⁴PBLs.

The ELISPOT assay represents a strong tool to monitor IDO peptidespecific T-cell responses. A major implication of the findings herein isthat the peptides of the invention are expressed and complexed with HLAmolecules on cancer cells and/or IDO expressing APCs. This renders thesecancer cells susceptible to destruction by CTLs and emphasizes thepotential usefulness of IDO immunization to fight cancer and infections.The presence of spontaneous CTL-responses in PBLs from melanoma patientsto HLA-restricted IDO derived peptide epitopes shows theimmunotherapeutic potential of IDO immunogenic peptides.

In an embodiment of the present invention the peptide of the inventionis capable of eliciting INF-γ-producing cells in a PBL population of anindividual suffering from an clinical condition where IDO of SEQ ID NO:(1, 13, 14, 15, and/or 16) or a functional homologue thereof having atleast 70% identity to SEQ ID NO 1 is expressed. The clinical conditionis preferably a cancer or and infection and most preferably a cancer.

Origin

The peptides of the invention are, as mentioned above, derived from IDOof SEQ ID NO: 1, 13, 14, 15, and/or 16 or a fragment hereof, morepreferably, the peptides are derived from IDO of SEQ ID NO: 1 and/or 16;and most preferably, the peptides are derived from IDO of SEQ ID NO: 1.The protein from which the peptide can be derived can be any IDO fromany animal species in which the protein is expressed. In preferredembodiments, the starting protein is from a mammalian species includinga rodent species, rabbit and a primate species such as humans. Based onthe sequence of the selected protein, the peptide of the invention isderived by any appropriate chemical or enzymatic treatment of theprotein starting material that results in a peptide of a suitable sizeas indicated above, or it can be synthesized by any conventional peptidesynthesis procedures with which the person of ordinary skills in the artis familiar. Most preferably, the IDO protein, protein fragment,peptide, variant, and/or functional homologues of any of the se arederived from IDO as the sequence of the protein is expressed in humans.

Individual

The individual to be treated with the vaccine composition of the presentinvention is an individual suffering from a clinical condition. Theindividual is preferably of a mammalian species and most preferably ahuman being. The individual may be of any age, young or old, and may beeither male or female. The clinical condition from which the individualsuffers may be a neoplastic disease such as a cancer, or an infectionsuch as a microbial or viral infection e.g. HIV.

An embodiment of the present invention provides a vaccine for thetreatment, reduction of risk of, stabilization of or prevention of acancer. In another embodiment the present invention provides a vaccinefor the treatment, reduction of risk of, stabilization of or preventionof a disease stemming from an infection, such as a microbial or viralinfection.

A further embodiment regards a vaccine composition comprising IDO of SEQID NO: (1, 13, 14, 15 and/or 16) or a functional homologue thereofhaving at least 70% identity to SEQ ID NO: (1, 13, 14, 15 and/or 16) oran immunogenically active peptide fragment comprising a consecutivesequence of said IDO or said functional homologue thereof or a nucleicacid encoding said IDO or said peptide fragment; and an adjuvant for thetreatment of a clinical condition characterized by the expression ofIDO.

Cancer

The vaccine composition of the present invention may be used to prevent,reduce the risk from or treat a clinical condition. Preferably, theclinical condition is associated with or characterized by the expressionof IDO. IDO may be IDO as identified in any of SEQ ID NOs: (1, 13, 14,15, and/or 16) and may be a homolog sharing at least 70% identity withany of these in their wild type forms, but need not be functional. It isunderstood hereby that the expression level of IDO (the expression beingexpression of hnRNA, mRNA, precursor protein, fully processed proteinand so on) is the same or higher than in an individual not sufferingfrom a clinical condition. In a preferred embodiment of the invention,the clinical condition is cancer. Cancer (malignant neoplasm) is a classof diseases in which a group of cells display the traits of uncontrolledgrowth (growth and division beyond the normal limits), invasion(intrusion on and destruction of adjacent tissues), and sometimesmetastasis (spread to other locations in the body via lymph or blood).These three malignant properties of cancers differentiate them frombenign tumors, which are self-limited, do not invade or metastasize.Most cancers form a tumor but some, like leukemia, do not. The term“cancer” as used herein is meant to encompass any cancer, neoplastic andpreneoplastic disease.

A non-limiting group of cancers given as examples of cancers that may betreated, managed and/or prevented by administration of the vaccine ofthe present invention include: colon carcinoma, breast cancer,pancreatic cancer, ovarian cancer, prostate cancer, fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangeosarcoma, lymphangeoendotheliasarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma,rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,papillary carcinoma, papillary adenocarcinomas, cystandeocarcinoma,medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonalcarcinoma, Wilms' tumor, cervical cancer, testicular tumor, lungcarcinoma, small cell lung carcinoma, bladder carcinoma, epithelialcarcinoma, glioblastomas, neuronomas, craniopharingiomas, schwannomas,glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma,pinealoma, hemangioblastoma, acoustic neuroama, oligodendroglioma,meningioma, melanoma, neuroblastoma, retinoblastoma, leukemias andlymphomas, acute lymphocytic leukemia and acute myelocytic polycythemiavera, multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chaindisease, acute nonlymphocytic leukemias, chronic lymphocytic leukemia,chronic myelogenous leukemia, Hodgkin's Disease, non-Hodgkin'slymphomas, rectum cancer, urinary cancers, uterine cancers, oralcancers, skin cancers, stomach cancer, brain tumors, liver cancer,laryngeal cancer, esophageal cancer, mammary tumors, childhood-nullacute lymphoid leukemia (ALL), thymic ALL, B-cell ALL, acute myeloidleukemia, myelomonocytoid leukemia, acute megakaryocytoid leukemia,Burkitt's lymphoma, acute myeloid leukemia, chronic myeloid leukemia,and T cell leukemia, small and large non-small cell lung carcinoma,acute granulocytic leukemia, germ cell tumors, endometrial cancer,gastric cancer, cancer of the head and neck, chronic lymphoid leukemia,hairy cell leukemia and thyroid cancer.

In a preferred embodiment the vaccine composition according to theinvention vaccine composition is capable of eliciting a clinicalresponse in subject, wherein the clinical response may be characterizedby a stable disease, in a preferred embodiment the clinical response maybe characterized by a partial response or preferably the clinicalresponse may be characterized by complete remission of a cancer.Preferably, the cancer is selected from the group of; melanoma, breastcancer, ovarian cancer, lung cancer, pancreatic cancer, hematologiccancers (such as leukemias), colon and renal cell cancers.

In one aspect of the invention the vaccine composition is capable ofeliciting a clinical response in an individual. In one embodiment theclinical response may be characterized by a stable disease (no furtherworsening or progression), in a preferred embodiment the clinicalresponse may be characterized by a partial response or preferably theclinical response may be characterized by complete remission of a canceror infections. The clinical response may be determined as describedherein below.

In another aspect of the invention the vaccine composition is capable ofeliciting a clinical response in subject, wherein the clinical responseis characterized by a decrease in the sum of the longest diameter of thelargest target lesion. The decrease may be determined as describedherein below.

All measurable lesions up to a maximum of five lesions per organ and 10lesions in total, representative of all involved organs should beidentified as target lesions and recorded and measured at baseline.

-   -   Target lesions should be selected on the basis of their size        (lesions with the longest diameter) and their suitability for        accurate repeated measurements (either by imaging techniques or        clinically).    -   A sum of the longest diameter (LD) for all target lesions will        be calculated and reported as the baseline sum LD. The baseline        sum LD will be used as reference by which to characterize the        objective tumor.    -   All other lesions (or sites of disease) should be identified as        non-target lesions and should also be recorded at baseline.        Measurements of these lesions are not required, but the presence        or absence of each should be noted throughout follow-up.

Evaluation of Target Lesions

-   -   Complete Response (CR): Disappearance of all target lesions    -   Partial Response (PR): At least a 30% decrease in the sum of the        LD of target lesions, taking as reference the baseline sum LD    -   Progressive Disease (PD): At least a 20% increase in the sum of        the LD of target lesions, taking as reference the smallest sum        LD recorded since the treatment started or the appearance of one        or more new lesions    -   Stable Disease (SD): Neither sufficient shrinkage to qualify for        PR nor sufficient increase to qualify for PD, taking as        reference the smallest sum LD since the treatment started

Evaluation of Non-Target Lesions

-   -   Complete Response (CR): Disappearance of all non-target lesions        and normalization of tumor marker level    -   Incomplete Response/Stable Disease (SD): Persistence of one or        more non-target lesion(s) or/and maintenance of tumor marker        level above the normal limits    -   Progressive Disease (PD): Appearance of one or more new lesions        and/or unequivocal progression of existing non-target lesions

In an embodiment of the present invention the vaccine compositioncomprising any of the herein mentioned proteins and/or polypeptides iscapable of eliciting a clinical response in subject, wherein theclinical response is characterized by a decrease in the sum of thelongest diameter of the largest target lesion

It is contemplated that the vaccine composition of the invention iscapable of eliciting an immune response against a cancer expressing IDOof SEQ ID NO: 1 or a functional homologue thereof having at least 70%identity to SEQ ID NO: 1, when administered to an individual sufferingfrom a cancer expressing IDO. The vaccine composition of the inventionis capable of eliciting the production in a vaccinated individual ofeffector T-cells having a cytotoxic effect against the cancer cells, IDOexpressing APCs and/or inducing infiltration of antigen specific T-cellsin tumor stroma in a subject.

In addition to their capacity to elicit immune responses in PBLpopulations it is also contemplated that the peptides of the inventionare capable of eliciting cytolytic immune responses in situ, i.e. insolid tumor tissues. This may for example be demonstrated by providingHLA-peptide complexes, e.g. being multimerized and being provided with adetectable label, and using such complexes for immunohistochemistrystainings to detect in a tumor tissue CTLs that are reactive with theepitope peptide of the invention. Accordingly, a further significantfeature of the peptide of the invention is that it is capable of in situdetection in a tumor tissue of CTLs that are reactive with the epitopepeptide.

It is also contemplated that the peptides of the invention, in additionto their capacity to bind to HLA molecules resulting in the presentationof complexes of HLA and peptides on cell surfaces, which complexes inturn act as epitopes or targets for cytolytic T cells, may elicit othertypes of immune responses, such as B-cell responses resulting in theproduction of antibodies against the complexes and/or a Delayed TypeHypersensitivity (DTH) reaction. The latter type of immune response isdefined as a redness and palpable induration at the site of injection ofthe peptide of the invention.

It is an object of the presenting invention to provide a vaccinecomposition comprising Indoleamine 2,3-dioxygenase (IDO) of SEQ ID NO:(1, 13, 14, 15 and/or 16) or a functional homologue thereof having atleast 70% identity to SEQ ID NO: (1, 13, 14, 15 and/or 16) or animmunogenically active peptide fragment comprising a consecutivesequence of said IDO or said functional homologue thereof or a nucleicacid encoding said IDO or said peptide fragment; and an adjuvant, forthe prevention of, reduction of risk from or treatment of cancer.

Cancer Combination Treatment

In some cases it will be appropriate to combine the treatment method ofthe invention with a further conventional cancer treatment such aschemotherapy, radiotherapy, treatment with immunostimulating substances,gene therapy, treatment with antibodies and treatment using dendriticcells.

Since elevated expression of IDO in tumor cells leads to inhibition ofthe immune system, the combination of a IDO-based immunotherapy asdisclosed by the present invention with cytotoxic chemotherapy and oranother anti-cancer immunotherapeutic treatment is an effective approachto treat cancer. These remedies are also referred to herein as “secondactive ingredients”.

Examples of chemotherapeutic agents that are of relevance in regards toco-administration (sequentially or simultaneously) with the vaccinecomposition of the present invention include, but are not limited to:all-trans retinoic acid, Actimide, Azacitidine, Azathioprine, Bleomycin,Carboplatin, Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide,Cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin,Epirubicin, Etoposide, Fludarabine, Fluorouracil, Gemcitabine,Hydroxyurea, Idarubicin, Irinotecan, Lenalidomide, Leucovorin,Mechlorethamine, Melphalan, Mercaptopurine, Methotrexate, Mitoxantrone,Oxaliplatin, Paclitaxel, Pemetrexed, Revlimid, Temozolomide, Teniposide,Thioguanine, Valrubicin, Vinblastine, Vincristine, Vindesine andVinorelbine. In one embodiment, a chemotherapeutic agent for use in thecombination of the present agent may, itself, be a combination ofdifferent chemotherapeutic agents. Suitable combinations include FOLFOXand IFL. FOLFOX is a combination which includes 5-fluorouracil (5-FU),leucovorin, and oxaliplatin. IFL treatment includes irinotecan, 5-FU,and leucovorin.

Another second active ingredient may be a kinase inhibitor, forseparate, simultaneous or combined use in the treatment of tumors.Suitable kinase inhibitors include those which have been shown topossess anti-tumor activity (such as gefitinib (Iressa) and erlotinib(Tarceva) and these could be used in combination with the peptides. Thereceptor tyrosine kinase inhibitors, such as Sunitinib malate andSorafenib which have been shown to be effective in the treatment ofrenal cell carcinoma are also suitable to be used as second activeingredients.

Further examples of second active ingredients are immunostimulatingsubstances e.g. cytokines and antibodies. Such as cytokines may beselected from the group consisting of, but not limited to: GM-CSF, typeI IFN, interleukin 21, interleukin 2, interleukin 12 and interleukin 15.The antibody is preferably an immunostimulating antibody such asanti-CD40 or anti-CTLA-4 antibodies. The immunostimulatory substance mayalso be a substance capable of depletion of immune inhibitory cells(e.g. regulatory T-cells) or factors, said substance may for example beE3 ubiquitin ligases. E3 ubiquitin ligases (the HECT, RING and U-boxproteins) have emerged as key molecular regulators of immune cellfunction, and each may be involved in the regulation of immune responsesduring infection by targeting specific inhibitory molecules forproteolytic destruction. Several HECT and RING E3 proteins have now alsobeen linked to the induction and maintenance of immune self-tolerance:c-Cbl, Cbl-b, GRAIL, Itch and Nedd4 each negatively regulate T cellgrowth factor production and proliferation.

In an embodiment, the vaccine composition of the present invention,comprising an IDO derived polypeptide, is administered in combinationwith a second active ingredient, such as an immunostimulatory substance.The immunostimulatory substance is preferably an interleukin such asIL-21 or IL-2 or a chemotherapeutic agent.

Infections

The word infection as used herein relates to any kind of clinicalcondition giving rise to an immune response, such as an inflammation,and therefore includes infectious diseases, chronic infections,autoimmune conditions and allergic inflammations. Thus, infections, suchas infectious diseases, chronic infections, autoimmune conditions andallergic inflammations are all clinical conditions of relevance for thepresent invention, and are dealt with in turn hereunder. Furthermore,the terms infection and inflammation are used interchangeably herein.

Inflammation is the complex biological response of vascular tissues toharmful stimuli, such as pathogens, damaged cells, or irritants. It is aprotective attempt by the organism to remove the injurious stimuli aswell as initiate the healing process for the tissue. Inflammation can beclassified as either acute or chronic. Acute inflammation is the initialresponse of the body to harmful stimuli and is achieved by the increasedmovement of plasma and leukocytes from the blood into the injuredtissues. A cascade of biochemical events propagates and matures theinflammatory response, involving the local vascular system, the immunesystem, and various cells within the injured tissue. Prolongedinflammation, known as chronic inflammation, leads to a progressiveshift in the type of cells which are present at the site of inflammationand is characterized by simultaneous destruction and healing of thetissue from the inflammatory process. In either case, IDO is expressedby cells of the immune system such as the APCs and therefore infectionsand inflammations are clinical conditions that may be treated,prevented, or from which the risk may be reduced by the administrationof the vaccine composition of the present invention. The vaccinecomposition preferably comprises IDO protein, protein fragments,polypeptide or peptides derived there from or functional homologues ofany of these.

Examples of disorders associated with inflammation which are ofrelevance to the presenting invention include, but are not limited to:Allergic inflammations, Asthma, Autoimmune diseases, Chronicinflammations, Chronic prostatitis, Glomerulonephritis,Hypersensitivities, Infectious diseases, Inflammatory bowel diseases,Pelvic inflammatory disease, Reperfusion injury, Rheumatoid arthritis,Transplant rejection, and Vasculitis.

Chronic Infections and Inflammations

Chronic inflammation is especially of relevance in regards to thepresent invention. A chronic inflammation is a pathological conditioncharacterized by concurrent active inflammation, tissue destruction, andattempts at repair. Chronically inflamed tissue is characterized by theinfiltration of mononuclear immune cells (monocytes, macrophages,lymphocytes, and plasma cells), tissue destruction, and attempts athealing, which include angiogenesis and fibrosis.

In acute inflammation, removal of the stimulus halts the recruitment ofmonocytes (which become macrophages under appropriate activation) intothe inflamed tissue, and existing macrophages exit the tissue vialymphatics. However in chronically inflamed tissue the stimulus ispersistent, and therefore recruitment of monocytes is maintained,existing macrophages are tethered in place, and proliferation ofmacrophages is stimulated (especially in atheromatous plaques).

It is an object of the presenting invention to provide a vaccinecomposition comprising Indoleamine 2,3-dioxygenase (IDO) of SEQ ID NO:(1, 13, 14, 15 and/or 16) or a functional homologue thereof having atleast 70% identity to SEQ ID NO: (1, 13, 14, 15 and/or 16) or animmunogenically active peptide fragment comprising a consecutivesequence of said IDO or said functional homologue thereof or a nucleicacid encoding said IDO or said peptide fragment; and an adjuvant, forthe prevention of, reduction of risk from or treatment of chronicinflammations.

Infectious Diseases

The vaccine composition of the present invention may be used to prevent,reduce the risk from or treat a clinical condition. In a preferredembodiment of the invention, the clinical condition is an infectiousdisease. The infectious disease may be promoted by any infectious agentsuch as bacteria, virus, parasites and or fungi that are capable ofinducing an increased expression of IDO in the individual suffering fromthe infectious disease, preferably, the infectious disease is or is atrisk of becoming a chronic disease. As described in the background ofinvention, the increased expression of IDO has an immediate effect onthe microbial agents in the vicinity of the IDO expressing organism bydepriving it of tryptophan. However, this approach backfires, as theincreased IDO expression induces inhibits the activity of Treg cells, ifthe IDO expressing cell is an APC. Therefore it is an aspect of thepresent invention to provide a vaccine composition comprising IDOprotein, protein fragments, peptides and or variant of any of these forthe treatment, amelioration of (lessening of severity) stabilizationand/or prevention of a disease caused by an infectious agent.

An infectious diseases may be caused by a virus, and viral diseasesagainst which the vaccine composition of the present invention may beadministered in the treatment of include, but are not limited to thefollowing viral diseases: HIV, AIDS, AIDS Related Complex, Chickenpox(Varicella), Common cold, Cytomegalovirus Infection, Colorado tickfever, Dengue fever, Ebola hemorrhagic fever, Hand, foot and mouthdisease, Hepatitis, Herpes simplex, Herpes zoster, HPV (Humanpapillomavirus), Influenza (Flu), Lassa fever, Measles, Marburghemorrhagic fever, Infectious mononucleosis, Mumps, Norovirus,Poliomyelitis, Progressive multifocal leukencephalopathy, Rabies,Rubella, SARS, Smallpox (Variola), Viral encephalitis, Viralgastroenteritis, Viral meningitis, Viral pneumonia, West Nile disease,and Yellow fever. Preferably, the vaccine composition is administered toindividuals suffering from HIV/AIDS and viral infections that may causecancer. The main viruses associated with human cancers are humanpapillomavirus, hepatitis B and hepatitis C virus, Epstein-Barr virus,and human T-lymphotropic virus; thus it is an object of the presentinvention to be administered as the treatment of or as part of thetreatment of these viral infections.

Examples of bacterial infections of relevance for the present inventioninclude, but are not limited to: Anthrax, Bacterial Meningitis,Botulism, Brucellosis, Campylobacteriosis, Cat Scratch Disease, Cholera,Diphtheria, Epidemic Typhus, Gonorrhea, Impetigo, Legionellosis, Leprosy(Hansen's Disease), Leptospirosis, Listeriosis, Lyme disease,Melioidosis, Rheumatic Fever, MRSA infection, Nocardiosis, Pertussis(Whooping Cough), Plague, Pneumococcal pneumonia, Psittacosis, Q fever,Rocky Mountain Spotted Fever (RMSF), Salmonellosis, Scarlet Fever,Shigellosis, Syphilis, Tetanus, Trachoma, Tuberculosis, Tularemia,Typhoid Fever, Typhus, and Urinary Tract Infections. It is an object ofthe present invention to provide a vaccine for the treatment and/orprevention and/or reduction of risk from a bacterial infection.

It is a further aspect of the present invention to provide a vaccinecomposition for the treatment and/or prevention and/or reduction of riskfrom: Parasitic infectious diseases such as, but not limited to: Africantrypanosomiasis, Amebiasis, Ascariasis, Babesiosis, Chagas Disease,Clonorchiasis, Cryptosporidiosis, Cysticercosis, Diphyllobothriasis,Dracunculiasis, Echinococcosis, Enterobiasis, Fascioliasis,Fasciolopsiasis, Filariasis, Free-living amebic infection, Giardiasis,Gnathostomiasis, Hymenolepiasis, Isosporiasis, Kala-azar, Leishmaniasis,Malaria, Metagonimiasis, Myiasis, Onchocerciasis, Pediculosis, PinwormInfection, Scabies, Schistosomiasis, Taeniasis, Toxocariasis,Toxoplasmosis, Trichinellosis, Trichinosis, Trichuriasis,Trichomoniasis, and Trypanosomiasis; Fungal infectious diseases such asbut not limited to: Aspergillosis, Blastomycosis, Candidiasis,Coccidioidomycosis, Cryptococcosis, Histoplasmosis, Tinea pedis; Prioninfectious diseases such as but not limited to: transmissible spongiformencephalopathy, Bovine spongiform encephalopathy, Creutzfeldt-Jakobdisease, Kuru-Fatal Familial Insomnia, and Alpers Syndrome; thus it isan object of the present invention to be administered as the treatmentof or as part of the treatment of these parasitic, fungal or prioncaused infections.

Infectious Disease Combination Treatment

It is further provided for that a treatment of any infectious disease bythe administration of the vaccine composition according to the presentinvention may be given in conjunction with a further (second) activeingredient or in combination with a further treatment such as antibiotictreatment, chemotherapy, treatment with immunostimulating substances,treatment using dendritic cells, antiviral agents anti parasitic agentsand so forth.

Examples of a second active ingredient that may be used in the treatmentof an infectious disease in combination with the vaccine of the presentinvention include, and are not limited to antibiotics. The termantibiotics herein refers to substances with anti-bacterial,anti-fungal, anti-viral and/or anti-parasitical activity; examples ofrelevance to the present invention include, but are not limited to:Amikacin, Gentamycin, Kanamycin, Neomycin, Netilmicin, Paromomycin,Streptomycin, Tobramycin, Ertapenem, Imipenem, Meropenem,Chloramphenicol, Fluoroquinolones, Ciprofloxacin, Gatifloxacin,Gemifloxacin, Grepafloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin,Norfloxacin, Ofloxacin, Sparfloxacin, Trovafloxacin, Glycopeptides,Vancomycin, Lincosamides, Clindamycin, Macrolides/Ketolides,Azithromycin, Clarithromycin, Dirithromycin, Erythromycin, Cefadroxil,Cefazolin, Cephalexin, Cephalothin, Cephapirin, Cephradine, Cefaclor,Cefamandole, Cefonicid, Cefotetan, Cefoxitin, Cefprozil, Cefuroxime,Loracarbef, Cefdinir, Cefditoren, Cefixime, Cefoperazone, Cefotaxime,Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftriaxone,Cefepime, Monobactams, Aztreonam, Nitroimidazoles, Metronidazole,Oxazolidinones, Linezolid, Penicillins, Amoxicillin,Amoxicillin/Clavulanate, Ampicillin, Sulbactam, Bacampicillin,Carbenicillin, Cloxacillin, Dicloxacillin, Methicillin, Mezlocillin,Nafcillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin,Piperacillin/Tazobactam, Ticarcillin, Ticarcillin/Clavulanate,Streptogramins, Quinupristin, Dalfopristin,Sulfonamide/Sulfamethoxazole, Trimethoprim, Tetracyclines,Demeclocycline, Doxycycline, Minocycline, Tetracycline, Azoleantifungals Clotrimazole Fluconazole, Itraconazole, Ketoconazole,Miconazole, Voriconazole, Amphotericin B, Nystatin, Echinocandin,Caspofungin, Micafungin, Ciclopirox, Flucytosine, Griseofulvin, andTerbinafine. Of further relevance are antivirals such as Vidarabine,Acyclovir, Gancyclovir and Valcyte (valganciclovir), Nucleoside-analogreverse transcriptase inhibitors (NRTI): AZT (Zidovudine), ddl(Didanosine), ddC (Zalcitabine), d4T (Stavudine), 3TC (Lamivudine),Non-nucleoside reverse transcriptase inhibitors (NNRTI): Nevirapine,Delavirdine, Protease Inhibitors: Saquinavir, Ritonavir, Indinavir,Nelfinavir, Ribavirin, Amantadine/Rimantadine, Relenza and Tamiflu,Pleconaril, Interferons

In an embodiment, the present invention regards a vaccine compositioncomprising IDO derived proteins, polypeptides and/or functional homologsof these for the treatment of an infectious disease in combination withat least one antibiotic. Preferably, the vaccine composition of thepresent invention is used for the treatment of chronic infections e.g.HIV and therefore is used in combination with any of the above listedantibiotics such as anti-viral agents.

Autoimmune Diseases

Autoimmune diseases arise when an organism fails to recognize its ownconstituent parts (down to the sub-molecular levels) as self, whichresults in an immune response against its own cells and tissues. Anydisease that results from such an aberrant immune response is termed anautoimmune disease and is of relevance to the present invention.Examples hereof include but are not limited to: Coeliac disease,diabetes mellitus type 1 (IDDM), systemic lupus erythematosus (SLE),Sjögren's syndrome, multiple sclerosis (MS), Hashimoto's thyroiditis,Graves' disease, idiopathic thrombocytopenic purpura, and rheumatoidarthritis (RA).

It is an object of the present invention to provide a vaccinecomposition comprising Indoleamine 2,3-dioxygenase (IDO) of SEQ ID NO:(1, 13, 14, 15 and/or 16) or a functional homologue thereof having atleast 70% identity to SEQ ID NO: (1, 13, 14, 15 and/or 16) or animmunogenically active peptide fragment comprising a consecutivesequence of said IDO or said functional homologue thereof or a nucleicacid encoding said IDO or said peptide fragment; and an adjuvant, forthe prevention of, reduction of risk from or treatment of autoimmunediseases.

Autoimmune Disease Combination Treatment

Current treatments for autoimmune disease are usually immunosuppressive,anti-inflammatory, or palliative. Non-immune therapies, such as hormonereplacement in Hashimoto's thyroiditis or diabetes mellitus Type 1treatment outcomes of the autoaggressive response. Dietary manipulationlimits the severity of celiac disease. Steroidal or NSAID treatmentlimits inflammatory symptoms of many diseases. Intravenous preparationsof immune globulin (IVIG) are used for Chronic InflammatoryDemyelinating Polyneuropathy (CIDP) and Guillain-Barré syndrome (GBS).More specific immunomodulatory therapies, such as the TNFα antagonistEtanercept, have been shown to be useful in treating RA. Theseimmunotherapies may be associated with increased risk of adverseeffects, such as susceptibility to infection.

Helminthic therapy has developed based on these observations andinvolves inoculation of the individual with specific parasiticintestinal nematodes (helminths). There are currently twoclosely-related treatments available, inoculation with either Necatoramericanus, commonly known as hookworms, or Trichuris Suis Ova, commonlyknown as Pig Whipworm Eggs. Research is available that demonstrates thisapproach is highly effective in treating a variety of autoimmunedisorders, including Crohn's, Ulcerative Colitis, Asthma, allergies,Multiple Sclerosis, and chronic inflammatory disorders

In an embodiment, the vaccine herein disclosed is used in combinationwith a second active ingredient such as any of the above mentioned drugsand treatments against autoimmune diseases.

Allergic Inflammation

Allergy is a disorder of the immune system often also referred to asatopy. Allergic reactions occur to environmental substances known asallergens; these reactions are acquired, predictable and rapid.Strictly, allergy is one of four forms of hypersensitivity and is calledtype I (or immediate) hypersensitivity. It is characterized by excessiveactivation of certain white blood cells called mast cells and basophilsby a type of antibody, known as IgE, resulting in an extremeinflammatory response. Common allergic reactions include eczema, hives,hay fever, asthma, food allergies, and reactions to the venom ofstinging insects such as wasps and bees.

Allergic inflammation is an important pathophysiological feature ofseveral disabilities or medical conditions including allergic asthma,atopic dermatitis, allergic rhinitis and several ocular allergicdiseases.

It is an object of the present invention to provide a vaccinecomposition comprising Indoleamine 2,3-dioxygenase (IDO) of SEQ ID NO:(1, 13, 14, 15 and/or 16) or a functional homologue thereof having atleast 70% identity to SEQ ID NO: (1, 13, 14, 15 and/or 16) or animmunogenically active peptide fragment comprising a consecutivesequence of said IDO or said functional homologue thereof or a nucleicacid encoding said IDO or said peptide fragment; and an adjuvant, forthe prevention of, reduction of risk from or treatment of allergicinflammation.

Allergic Inflammation Combination Treatment

Two types of treatments are available for the treatment of allergicinflammations, pharmacotherapy and immunotherapy: pharmacotherapy andimmunotherapy.

Pharmacotherapy, is the use of antagonistic drugs to block the action ofallergic mediators, or to prevent activation of cells and degranulationprocesses. These include antihistamines, cortisone, dexamethasone,hydrocortisone, epinephrine (adrenaline), theophylline, cromolyn sodiumand anti-leukotrienes, such as Montelukast (Singulair) or Zafirlukast(Accolate); anti-cholinergics, decongestants, mast cell stabilizers, andother compounds thought to impair eosinophil chemotaxis, are alsocommonly used. Immunotherapy is the desensitization or hyposensitizationtreatment in which the individual is gradually vaccinated withprogressively larger doses of the allergen in question. A second form ofimmunotherapy involves the intravenous injection of monoclonal anti-IgEantibodies. A third type, Sublingual immunotherapy, is anorally-administered therapy which takes advantage of oral immunetolerance to non-pathogenic antigens such as foods and residentbacteria.

In an embodiment, the vaccine herein disclosed is used in combinationwith a second active ingredient such as any of the above mentioned drugsand treatments against allergic inflammations.

Pharmaceutical Compositions

The present invention regards pharmaceutical compositions capable oftreating, reducing the risk of and/or preventing a clinical disorderassociated with IDO expression in an individual; in other words theterms vaccine and pharmaceutical composition are used interchangeablyherein. The vaccine/pharmaceutical compositions of the present inventionmay be “traditional” vaccine compositions comprising antigens such asproteins polypeptides and/or nucleic acid molecules. They may also be inthe form of compositions comprising cells, such as modified cellsoriginating from the individual and later processed, or to compositionscomprising complex molecules such as antibodies or TCRs.

Generally, a vaccine is a substance or composition capable of inducingan immune response in an individual. The composition may comprise one ormore of the following: an “active component” such as an antigen(s) (e.g.protein, polypeptides, peptides, nucleic acids and the like), nucleicacid constructs comprising one or more antigens amongst other elements,cells, (e.g. loaded APC, T cells for adoptive transder aso.), complexmolecules (Antibodies, TCRs and MHC complexes and more), carriers,adjuvants and pharmaceutical carriers. In the following, the variouscomponents of a vaccine composition according to the present inventionare disclosed in more detail.

The vaccine composition of the invention is capable of eliciting animmune response against a cancer, DC or APC expressing IDO of SEQ ID NO:1 or a functional homologue thereof having at least 70% identity to SEQID NO 1, when administered to an individual suffering from a cancerand/or infection (leading to the expression of IDO). In a preferredembodiment the clinical condition is a cancer. The vaccine compositionof the invention is capable of eliciting the production in a vaccinatedindividual of effector T-cells having a cytotoxic effect against cancercells, APCs and DCs expressing IDO and/or inducing infiltration ofantigen specific T-cells in tumor stroma in a subject.

Antigens and Other Active Components Protein/Polypeptide Based VaccineCompositions

The peptides of the present invention bind with surprisingly highaffinity (see FIG. 2) and are ready for use as antigens as they arepresented here. Preferably, the vaccine composition of the presentinvention comprises one or more of the following: IDO protein (SEQ IDNO: 1), polypeptide fragments here from, likewise variants, functionalhomologues of full length and partial length IDO, contiguous peptides ofIDO and functional homologues of these. More preferably, the vaccinecomposition comprises any of the sequences listed in the sequence listof the present disclosure. Very preferably, the vaccine compositioncomprises the peptides IDO5 (SEQ ID NO: 6), IDO 2 (SEQ ID NO: 3), and/orIDO6 (SEQ ID NO: 7).

The choice of antigen in the vaccine composition of the invention willdepend on parameters determinable by the person of skill in the art. Asit has been mentioned, each of the different peptides of the inventionis presented on the cell surfaces by a particular HLA molecule. As such,if a subject to be treated is typed with respect to HLA phenotype, apeptide/peptides are selected that is/are known to bind to thatparticular HLA molecule. Alternatively, the antigen of interest isselected based on the prevalence of the various HLA phenotypes in agiven population. As an example, HLA-A2 is the most prevalent phenotypein the Caucasian population, and therefore, a composition containing apeptide binding to HLA-A2 will be active in a large proportion of thatpopulation. Furthermore, the antigens/peptides of the present inventionmay be modified according to the anchor residue motifs presented inTable 2, to enhance binding to particular HLA molecules.

The composition of the invention may also contain a combination of twoor more IDO derived peptides, each interacting specifically with adifferent HLA molecule so as to cover a larger proportion of the targetpopulation. Thus, as examples, the pharmaceutical composition maycontain a combination of a peptide restricted by a HLA-A molecule and apeptide restricted by a HLA-B molecule, e.g. including those HLA-A andHLA-B molecules that correspond to the prevalence of HLA phenotypes inthe target population, such as e.g. HLA-A2 and HLA-B35. Additionally,the composition may comprise a peptide restricted by an HLA-C molecule.

In the case of peptide-based vaccines, epitopes can be administered inan ‘MHC-ready’ form, which enables presentation through exogenousloading independently of antigen uptake and processing by hostantigen-presenting cells. The peptides of the present invention compriseboth peptides in a short ‘MHC-ready’ form and in a longer form requiringprocessing by the proteasome thus providing a more complex vaccinecomposition that can target multiple tumor antigens. The more differentHLA groups are targeted by a vaccine, the higher likelihood of thevaccine functioning in diverse populations.

The present invention regards in a preferred embodiment a vaccinecomposition comprising Indoleamine 2,3-dioxygenase (IDO) of SEQ ID NO: 1or a functional homologue thereof having at least 70% identity to SEQ IDNO: 1 or an immunogenically active peptide fragment comprising aconsecutive sequence of said IDO or said functional homologue thereof ora nucleic acid encoding said IDO or said peptide fragment; incombination with an adjuvant for use as a medicament. The vaccinecomposition may be administered to treat, prevent, or reduce the riskassociated with a clinical condition in an individual.

Multi Epitope Vaccine Composition

The invention also relates to highly immunogenic multi-epitope vaccines.Preferably, such vaccines should be designed so as to facilitate asimultaneous delivery of the best-suited IDO-derived peptides optionallyin combination with other suitable peptides and/or adjuvants asdescribed hereinafter. The present invention encompasses suchmultiepitope vaccines comprising IDO-derived peptides optionally incombination with further proteins or peptides fragments not belonging toor derived from IDO and/or adjuvants as described hereinafter. Animportant factor driving the development of vaccines having a morecomplex composition is the desire to target multiple tumor antigens e.g.by designing vaccines comprising or encoding a collection of carefullyselected CTL and T_(h) cell epitopes. The invention thus in one aspectrelates to vaccine compositions comprising both Class I and ClassII-restricted IDO epitopes.

The peptides of the present invention thus comprise both peptides in ashort ‘MHC-ready’ form (class I restricted), and in a longer formrequiring processing by the proteasome (class II restricted). Thus, thecomposition according to the present invention may be provided as amultiepitope vaccine comprising class I restricted epitope and/or classII restricted epitopes as defined hereinbefore.

Nucleic Acid Based Vaccine Composition

The vaccine composition according to the present invention may comprisea nucleic acid encoding a protein belonging to the IDO or animmunologically active peptide fragment thereof. Said nucleic acid maythus encode any of the above-mentioned proteins and peptide fragments.The nucleic acid may for example be DNA, RNA, LNA, HNA, PNA, preferablythe nucleic acid is DNA or RNA.

The nucleic acids of the invention may be comprised within any suitablevector, such as an expression vector. Numerous vectors are available andthe skilled person will be able to select a useful vector for thespecific purpose. The vector may, for example, be in the form of aplasmid, cosmid, viral particle or artificial chromosome. Theappropriate nucleic acid sequence may be inserted into the vector by avariety of procedures, for example, DNA may be inserted into anappropriate restriction endonuclease site(s) using techniques well knownin the art. Apart from the nucleic acid sequence according to theinvention, the vector may furthermore comprise one or more of a signalsequence, an origin of replication, one or more marker genes, anenhancer element, a promoter, and a transcription termination sequence.The vector may also comprise additional sequences, such as enhancers,poly-A tails, linkers, polylinkers, operative linkers, multiple cloningsites (MCS), STOP codons, internal ribosomal entry sites (IRES) and hosthomologous sequences for integration or other defined elements. Methodsfor engineering nucleic acid constructs are well known in the art (see,e.g., Molecular Cloning: A Laboratory Manual, Sambrook et al., eds.,Cold Spring Harbor Laboratory, 2nd Edition, Cold Spring Harbor, N.Y.,1989). The vector is preferably an expression vector, comprising thenucleic acid operably linked to a regulatory nucleic acid sequencedirecting expression thereof in a suitable cell. Within the scope of thepresent invention said regulatory nucleic acid sequence should ingeneral be capable of directing expression in a mammalian cell,preferably a human cell, more preferably in an antigen presenting cell.

In one preferred embodiment the vector is a viral vector. The vector mayalso be a bacterial vector, such as an attenuated bacterial vector.Attenuated bacterial vectors may be used in order to induce lastingmucosal immune responses at the sites of infection and persistence.Different recombinant bacteria may be used as vectors, for example thebacterial vector may be selected from the group consisting ofSalmonella, Lactococcus], and Listeria. In general, induction ofimmunity to the heterologous antigen HPV16 L1 or E7 could be shown, withstrong CTL induction and tumor regression in mice. The vector mayfurthermore comprise a nucleic acid encoding a T-cell stimulatorypolypeptide.

Loaded APCs

In useful embodiments an immunogenic response directed against a cancerdisease is elicited by administering the peptide of the invention eitherby loading MHC class I or class II molecules on antigen presenting cells(APCs) from the individual, by isolating PBLs from the individual andincubating the cells with the peptide prior to injecting the cells backinto the individual or by isolating precursor APCs from the individualand differentiating the cells into professional APCs using cytokines andantigen before injecting the cells back into the individual.

It is thus an aspect of the invention to provide vaccine compositionscomprising antigen presenting cells comprising IDO or an immunologicallyactive peptide fragment thereof or a nucleic acid encoding said proteinor said immunologically active peptide fragment. The antigen presentingcell may be any cell capable of presenting an antigen to a T-cell.Preferred antigen presenting cells are dendritic cells. The dendriticcells (DC) may be prepared and used in therapeutic procedure accordingto any suitable protocol, for example as described herein below. It willbe appreciated by the person skilled in the art that the protocol may beadopted to use with individuals with different HLA type and differentdiseases.

Dendritic cells (DC) may be pulsed with 50 μg/ml HLA-restricted peptide(synthesized at GMP quality) for 1 h at 37° C. peptide and 5×10⁶ cellsare administered subcutaneously at day 1 and 14, subsequently every 4weeks, additional leukapheresis after 5 vaccinations. The generation ofDC for clinical use and quality control can be performed essentially asdescribed in Nicolette et al., (2007).

Thus, in one embodiment of the present invention, a method for treatingan individual suffering from a clinical condition characterized by theexpression of IDO, preferably wherein the clinical condition is canceror an infection, is one wherein the peptide is administered bypresenting the peptide to the individual's antigen presenting cells(APCs) ex vivo followed by injecting the thus treated APCs back into theindividual. There are at least two alternative ways of performing this.One alternative is to isolate APCs from the individual and incubate(load) the MHC class I molecules with the peptide. Loading the MHC classI molecules means incubating the APCs with the peptide so that the APCswith MHC class I molecules specific for the peptide will bind thepeptide and therefore be able to present it to T cells. Subsequently,the APCs are re-injected into the individual. Another alternative wayrelies on the recent discoveries made in the field of dendritic cellbiology. In this case, monocytes (being dendritic cell precursors) areisolated from the individual and differentiated in vitro intoprofessional APC (or dendritic cells) by use of cytokines and antigen.Subsequently, the in vitro generated DCs are pulsed with the peptide andinjected into the individual.

Adoptive Immunotherapy/Adoptive Transfer

An important aspect the invention relates to cultivating IDO specificT-cells in vitro and adoptive transfer of these to individuals. Adoptivetransfer means that the physician directly transfers the actualcomponents of the immune system that are already capable of producing aspecific immune response, into an individual.

It is one objective to the present invention to provide IDO specificT-cells, which may be useful for example for adoptive transfer. IsolatedT-cells comprising T-cell receptors capable of binding specifically toIDO peptide/MHC class I or IDO peptide/MHC class II complexes can beadoptively transferred to individuals, said T-cells preferably beingT-cells that have been expanded in vitro, wherein the IDO peptide may beany of the IDO peptides mentioned herein above. Methods of expandingT-cells in vitro are well known to the skilled person. The inventionalso relates to methods of treatment comprising administering T-cellscomprising T-cell receptors capable of binding specifically to a MHC−restricted IDO peptide complex to an individual, such as a human beingsuffering from a cancer disease, wherein the IDO derived peptide may beany of the IDO peptides mentioned herein above. The inventionfurthermore relates to use of T-cells comprising T-cell receptorscapable of binding specifically to IDO or peptide fragments thereof forthe preparation of a medicament for the treatment of a cancer orinfection. Autologous T-cell transfer may be performed essentially asdescribed in Walter et al., (1995).

TCR Transfer

In yet another embodiment, such T-cells could be irradiated beforeadoptive transfer to control proliferation in the individual. It ispossible to genetically engineer the specificity of T cells by TCR genetransfer (Engels et al., 2007). This allows the transfer of T cellsbearing IDO peptide specificity into individuals. In general, the use ofT cells for adoptive immunotherapy is attractive because it allows theexpansion of T cells in a tumor- or virus-free environment, and theanalysis of T cell function prior to infusion. The application of TCRgene-modified T cells (such as T-cells transformed with an expressionconstruct directing expressing of a heterologous TCR) in adoptivetransfer has several advantages in comparison to the transfer of T celllines: (i) the generation of redirected T cells is generally applicable.(ii) High-affinity or very high-affinity TCRs can be selected or createdand used to engineer T cells. (iii) High-avidity T cells can begenerated using codon optimized or murinized TCRs allowing bettersurface expression of the stabilized TCRs. Genetic engineering of T cellspecificity by T cell receptor (TCR) gene transfer may be performedessentially as described in Morgan et al., (2006).

TCR Transfection

TCR with known anti-tumor reactivity can be genetically introduced intoprimary human T lymphocytes. Genes encoding TCR alpha and beta chainsfrom a tumor specific CTL clone can be transfected into primary T cellsand in this way reprogram T cells with specificity against the tumorantigen. TCR RNA is transfected into PBL by electroporation (Schaft etal., 2006). Alternatively, T cells can be provided with at newspecificity by TCR gene transfer using retroviral vectors (Morgan etal., 2006). However, the provirus from the retroviral vector mightintegrate at random in the genome of the transfected cells andsubsequently disturb cell growth. Electroporation of T cells withTCR-coding RNA overcome this disadvantage, since RNA is only transientlypresent in the transfected cells and can not be integrated in the genome(Schaft et al., 2006). Furthermore, transfection of cells is routinelyused in the laboratory.

Adjuvants and Carriers

The vaccine composition according to the invention preferably comprisesan adjuvant and/or a carrier. Examples of useful adjuvants and carriersare given herein below. Thus the IDO protein, polypeptide fragment,variant or peptide derived here from may in a composition of the presentinvention be associated with an adjuvant and/or a carrier.

Adjuvants are any substance whose admixture into the vaccine compositionincreases or otherwise modifies the immune response to the IDO orpeptide fragment thereof, see further in the below. Carriers arescaffold structures, for example a polypeptide or a polysaccharide, towhich the IDO or peptide fragment thereof is capable of being associatedand which aids in the presentation of especially the peptides of thepresent invention.

Many of the peptides of the invention are relatively small molecules andit may therefore be required in compositions as described herein tocombine the peptides with various materials such as adjuvants and/orcarriers, to produce vaccines, immunogenic compositions, etc. Adjuvants,broadly defined, are substances which promote immune responses. Ageneral discussion of adjuvants is provided in Goding, MonoclonalAntibodies: Principles & Practice (2nd edition, 1986) at pages 61-63.Goding notes, that when the antigen of interest is of low molecularweight, or is poorly immunogenic, coupling to an immunogenic carrier isrecommended. Examples of such carrier molecules include keyhole limpethaemocyanin, bovine serum albumin, ovalbumin and fowl immunoglobulin.Various saponin extracts have also been suggested to be useful asadjuvants in immunogenic compositions. It has been proposed to usegranulocyte-macrophage colony stimulating factor (GM-CSF), a well knowncytokine, as an adjuvant (WO 97/28816).

A carrier may be present independently of an adjuvant. The function of acarrier can for example be to increase the molecular weight of inparticular peptide fragments in order to increase their activity orimmunogenicity, to confer stability, to increase the biologicalactivity, or to increase serum half-life. Furthermore, a carrier may aidin presenting the IDO protein, polypeptide, variant or peptide fragmentsthereof to T-cells. The carrier may be any suitable carrier known to aperson skilled in the art, for example a protein or an antigenpresenting cell. A carrier protein could be, but is not limited to,keyhole limpet hemocyanin, serum proteins such as transferrin, bovineserum albumin, human serum albumin, thyroglobulin or ovalbumin,immunoglobulins, or hormones, such as insulin or palmitic acid. Forimmunization of humans, the carrier must be a physiologically acceptablecarrier acceptable to humans and safe. However, tetanus toxoid and/ordiptheria toxoid are suitable carriers in one embodiment of theinvention. Alternatively, the carrier may be dextrans for examplesepharose.

Thus it is an aspect of the present invention that the IDO protein,polypeptide fragment, variant or peptide derived here from present inthe composition is associated with a carrier such as e.g. a protein ofthe above or an antigen-presenting cell such as e.g. a dendritic cell(DC).

Adjuvants could for example be selected from the group consisting of:AIK(SO₄)₂, AINa(SO₄)₂, AINH₄ (SO₄), silica, alum, AI(OH)₃, Ca₃ (PO₄)₂,kaolin, carbon, aluminum hydroxide, muramyl dipeptides,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP),N-acetyl-nornuramyl-L-alanyl-D-isoglutamine (CGP 11687, also referred toas nor-MDP),N-acetylmuramyul-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′2′-dipalmitoyl-sn-glycero-3-hydroxphosphoryloxy)-ethylamine(CGP 19835A, also referred to as MTP-PE), RIBI (MPL+TDM+CWS) in a 2%squalene/Tween-80® emulsion, lipopolysaccharides and its variousderivatives, including lipid A, Freund's Complete Adjuvant (FCA),Freund's Incomplete Adjuvants, Merck Adjuvant 65, polynucleotides (forexample, poly IC and poly AU acids), wax D from Mycobacterium,tuberculosis, substances found in Corynebacterium parvum, Bordetellapertussis, and members of the genus Brucella, Titermax, ISCOMS, Quil A,ALUN (see U.S. Pat. Nos. 58,767 and 5,554,372), Lipid A derivatives,choleratoxin derivatives, HSP derivatives, LPS derivatives, syntheticpeptide matrixes or GMDP, Interleukin 1, Interleukin 2, Montanide ISA-51and QS-21. Preferred adjuvants to be used with the invention includeoil/surfactant based adjuvants such as Montanide adjuvants (availablefrom Seppic, Belgium), preferably Montanide ISA-51. Other preferredadjuvants are bacterial DNA based adjuvants, such as adjuvants includingCpG oligonucleotide sequences. Yet other preferred adjuvants are viraldsRNA based adjuvants, such as poly I:C. Imidazochinilines are yetanother example of preferred adjuvants. The most preferred adjuvants areadjuvants suitable for human use.

Montanide adjuvants (all available from Seppic, Belgium), may beselected from the group consisting of Montanide ISA-51, MontanideISA-50, Montanide ISA-70, Montanide ISA-206, Montanide ISA-25, MontanideISA-720, Montanide ISA-708, Montanide ISA-763A, Montanide ISA-207,Montanide ISA-264, Montanide ISA-27, Montanide ISA-35, Montanide ISA51F, Montanide ISA 016D and Montanide IMS, preferably from the groupconsisting of Montanide ISA-51, Montanide IMS and Montanide ISA-720,more preferably from the group consisting of Montanide ISA-51. MontanideISA-51 (Seppic, Inc.) is oil/surfactant based adjuvants in whichdifferent surfactants are combined with a non-metabolizable mineral oil,a metabolizable oil, or a mixture of the two. They are prepared for useas an emulsion with an aqueous solution comprising IDO or peptidefragment thereof. The surfactant is mannide oleate. QS-21 (Antigenics;Aquila Biopharmaceuticals, Framingham, Mass.) is a highly purified,water-soluble saponin that handles as an aqueous solution. QS-21 andMontanide ISA-51 adjuvants can be provided in sterile, single-use vials.

The well-known cytokine GM-CSF is another preferred adjuvant of thepresent invention. GM-CSF has been used as an adjuvant for a decade andmay preferably be GM-CSF as described in WO 97/28816.

Desirable functionalities of adjuvants capable of being used inaccordance with the present invention are listed in the below table.

TABLE 2 Modes of adjuvant action Action Adjuvant type Benefit 1. Immuno-Generally small molecules or proteins Upregulation of immune response.modulation which modify the cytokine network Selection of Th1 or Th2 2.Presentation Generally amphipathic molecules or Increased neutralizingantibody complexes which interact with response. Greater duration ofimmunogen in its native conformation response 3. CTL induction Particleswhich can bind or Cytosolic processing of protein enclose immunogen andwhich yielding correct class 1 restricted can fuse with or disrupt cellpeptides membranes w/o emulsions for direct Simple process ifpromiscuous attachment of peptide to cell peptide(s) known surface MHC-14. Targeting Participate adjuvants which bind Efficient use of adjuvantand immunogen. Adjuvants which immunogen saturate Kupffer cellsCarbohydrate adjuvants which As above. May also determine target lectinreceptors on type of response if targeting macrophages and DCs selective5. Depot w/o emulsion for short term Efficiency Generation Microspheresor nanospheres for Potential for single-dose vaccine long term Source:Cox, J. C., and Coulter, A. R. (1997). Vaccine 15, 248-56.

A vaccine composition according to the present invention may comprisemore than one adjuvant. Furthermore, the invention encompasses atherapeutic composition further comprising any adjuvant substance and/orcarrier including any of the above or combinations thereof. It is alsocontemplated that the IDO protein, variants or peptide fragmentsthereof, and the adjuvant can be administered separately in anyappropriate sequence. Preferably, the vaccine compositions of thepresent invention comprise a Montanide adjuvant such as Montanide ISA 51or Montanide ISA 720 or the GM-CSF adjuvant.

Accordingly, the invention encompasses a therapeutic composition furthercomprising an adjuvant substance including any of the above orcombinations thereof. It is also contemplated that the antigen, i.e. thepeptide of the invention and the adjuvant can be administeredsimultaneously or separately in any appropriate sequence.

Doses and Administration

The amount of the immunogenic peptide of the invention in thepharmaceutical composition may vary, depending on the particularapplication. However, a single dose of the peptide composition ispreferably anywhere from about 10 μg to about 5000 μg, more preferablyfrom about 50 μg to about 2500 μg such as about 100 μg to about 1000 μg.Modes of administration include intradermal, subcutaneous andintravenous administration, implantation in the form of a time releaseformulation, etc. Any and all forms of administration known to the artare encompassed herein. Also any and all conventional dosage forms thatare known in the art to be appropriate for formulating injectableimmunogenic peptide composition are encompassed, such as lyophilizedforms and solutions, suspensions or emulsion forms containing, ifrequired, conventional pharmaceutically acceptable carriers, diluents,preservatives, adjuvants, buffer components, etc.

The pharmaceutical compositions may be prepared and administered usingany conventional protocol known by a person skilled in the art. Inexamples 3-5 non-limiting examples of preparation of a vaccinecomposition according to the invention is given as well as anon-limiting example of administration of such as a vaccine. It will beappreciated by the person skilled in the art that the protocol may beeasily adapted to any of the vaccine compositions described herein. In afurther embodiment of the invention, the pharmaceutical composition ofthe invention is useful for treating an individual suffering from aclinical condition characterized by expression of IDO, such as cancerand infections.

The immunoprotective effect of the composition of the invention can bedetermined using several approaches known to those skilled in the art. Asuccessful immune response may also be determined by the occurrence ofDTH reactions after immunization and/or the detection of antibodiesspecifically recognizing the peptide(s) of the vaccine composition.

Vaccine compositions according to the invention may be administered toan individual in therapeutically effective amounts. The effective amountmay vary according to a variety of factors such as the individual'scondition, weight, sex and age. Other factors include the mode ofadministration.

The pharmaceutical compositions may be provided to the individual by avariety of routes such as subcutaneous, topical, oral and intramuscular.Administration of pharmaceutical compositions is accomplished orally orparenterally. Methods of parenteral delivery include topical,intra-arterial (directly to the tissue), intramuscular, subcutaneous,intramedullary, intrathecal, intraventricular, intravenous,intraperitoneal, or intranasal administration. The present inventionalso has the objective of providing suitable topical, oral, systemic andparenteral pharmaceutical formulations for use in the methods ofprophylaxis and treatment with the vaccine composition.

For example, the vaccine compositions can be administered in such oraldosage forms as tablets, capsules (each including timed release andsustained release formulations), pills, powders, granules, elixirs,tinctures, solutions, suspensions, syrups and emulsions, or byinjection. Likewise, they may also be administered in intravenous (bothbolus and infusion), intraperitoneal, subcutaneous, topical with orwithout occlusion, or intramuscular form, all using forms well known tothose of ordinary skill in the pharmaceutical arts. An effective butnon-toxic amount of the vaccine, comprising any of the herein describedcompounds can be employed as a prophylactic or therapeutic agent. Alsoany and all conventional dosage forms that are known in the art to beappropriate for formulating injectable immunogenic peptide compositionare encompassed, such as lyophilized forms and solutions, suspensions oremulsion forms containing, if required, conventional pharmaceuticallyacceptable carriers, diluents, preservatives, adjuvants, buffercomponents, etc.

Preferred modes of administration of the vaccine composition accordingto the invention include, but are not limited to systemicadministration, such as intravenous or subcutaneous administration,intradermal administration, intramuscular administration, intranasaladministration, oral administration, rectal administration, vaginaladministration, pulmonary administration and generally any form ofmucosal administration. Furthermore, it is within the scope of thepresent invention that the means for any of the administration formsmentioned in the herein are included in the present invention.

A vaccine according to the present invention can be administered once,or any number of times such as two, three, four or five times.Administering the vaccine more than once has the effect of boosting theresulting immune response. The vaccine can further be boosted byadministering the vaccine in a form or body part different from theprevious administration. The booster shot is either a homologous or aheterologous booster shot. A homologous booster shot is a where thefirst and subsequent vaccinations comprise the same constructs and morespecifically the same delivery vehicle especially the same viral vector.A heterologous booster shot is where identical constructs are comprisedwithin different viral vectors.

Second Active Ingredient

It is an aspect of the present invention that the vaccine compositionherein provided is used in combination with a second active ingredient.The administration of the vaccine composition and the second activeingredient may be sequential or combined. Examples of second activeingredients are given above for both cancers and infections. It is afurther aspect that the vaccine composition may be used in combinationwith other therapy of relevance for the given clinical condition to betreated. Such therapy may include surgery, chemotherapy or gene therapy,immunostimulating substances or antibodies; a person skilled in the artis able to determine the appropriate combination treatment for a givenscenario.

In some cases it will be appropriate to combine the treatment method ofthe invention with a further medical treatment such as chemotherapy,radiotherapy, treatment with immunostimulating substances, gene therapy,treatment with antibodies and/or antibiotics and treatment usingdendritic cells.

Diagnostic and Prognostic Tools

The peptides of the present invention provide the basis for developingwidely applicable diagnostic and prognostic procedures in respect ofcancer diseases and infections. Thus, in other useful embodiments thecomposition of the invention is a composition for ex vivo or in situdiagnosis of the presence of IDO expressing cells in an individual. Thediagnostic procedure is based on the detection of IDO reactive T cellsamong PBLs or in tumor tissue.

Accordingly, there is provided a diagnostic kit for ex vivo or in situdiagnosis of the presence in an individual of IDO reactive T cells amongPBLs or in tumour tissue comprising one or more peptides of theinvention, and a method of detecting in an individual the presence ofsuch reactive T cells, the method comprising contacting a tumour tissueor a blood sample with a complex of a peptide of the invention and aClass I or Class II HLA molecule or a fragment of such molecule anddetecting binding of the complex to the tissue or the blood cells. Inone aspect, the invention provides a complex of a peptide of theinvention and a Class I or Class II HLA molecule or a fragment of suchmolecule, which is useful as a diagnostic reagent such as it isdescribed herein. Such a complex may be monomeric or multimeric.

Another useful diagnostic or prognostic approach is based on generatingantibodies in a heterologous animal species, e.g. murine antibodiesdirected against a human IDO-derived peptide of the invention, which canthen be used, e.g. to diagnose for the presence of cancer cellspresenting the peptide. For such immunization purposes, the amount ofpeptide may be less than that used in the course of in vivo therapy,such as that mentioned above. In general, a preferred dose can rangefrom about 1 μg to about 750 μg of peptide. It is also possible toproduce monoclonal antibodies based on immunization with a peptide ofthe invention. Accordingly, the present invention also relates to amolecule, in particular a monoclonal or polyclonal antibody including afragment hereof, that is capable of binding specifically to a peptide ofthe invention and to a molecule that is capable of blocking such abinding, e.g. an antibody raised against the monoclonal or polyclonalantibody directed against a peptide of the invention. The inventionfurthermore relates to isolated T-cell receptors capable of bindingspecifically to a peptide or a protein of the invention as well as toisolated nucleic acids encoding same. Such T-cell receptors may forexample be cloned from protein or peptide specific T-cells usingstandard techniques well known to the skilled person.

In one aspect the invention also relates to isolated T-cells comprisingT-cell receptors capable of binding specifically to IDO and/or peptidefragments thereof described herein. The isolated T-cells may be CD8T-cells or CD4 T-cells. The isolated T-cells are preferably T-cells thathave been expanded in vitro. Methods of expanding T-cells in vitro arewell known to the skilled person. Such T-cells may in particular beuseful in the treatment of cancer by adaptive transfer or autologouscell transfer. Thus, the invention also relates to pharmaceuticalcompositions comprising T-cells as well as methods of treatmentcomprising administering T-cells comprising T-cell receptors capable ofbinding specifically to IDO or peptide fragments thereof to anindividual, in need thereof such as an individual suffering from cancerand/or infections. Autologous cell transfer may be performed essentiallyas described in Walter et al., (1995).

The present invention provides the means for treating, preventing,alleviating or curing a clinical condition characterized by expressionof IDO such as cancers and infections preferably a cancer, comprisingadministering to an individual suffering from the disease an effectiveamount of a composition as defined herein, a molecule that is capable ofbinding specifically to a peptide fragment, which may for example be anantibody or a T-cell receptor or the kit-of-parts described herein.Accordingly, it is a further aspect of the invention to provide a methodof treating a clinical condition associated with the expression of IDOof SEQ ID NO: 1 and/or SEQ ID NO: 16.

Monitoring Immunization

In preferred embodiments, the pharmaceutical composition of theinvention is a vaccine composition. It is therefore of interest, and anaspect of the present invention to monitor the immunization in anindividual to whom the vaccine composition of the present invention isadministered. The pharmaceutical composition may thus be an immunogeniccomposition or vaccine capable of eliciting an immune response to acancer and/or infection. As used herein, the expression “immunogeniccomposition or vaccine” refers to a composition eliciting at least onetype of immune response directed against IDO expressing cells such ascancer cells, APCs or DCs. Thus, such an immune response may be any ofthe following: A CTL response where CTLs are generated that are capableof recognizing the HLA/peptide complex presented on cell surfacesresulting in cell lysis, i.e. the vaccine elicits the production in thevaccinated subject of effector T-cells having a cytotoxic effect againstthe cancer cells; a B-cell response giving rise to the production ofanti-cancer antibodies; and/or a DTH type of immune response. It is onobject of the present invention to monitor the immunization of anindividual by monitoring any of the above reactions subsequent toadministering the composition of the present invention to saidindividual.

In one aspect the invention relates to methods of monitoringimmunization, said method comprising the steps of

-   -   i) providing a blood sample from an individual    -   ii) providing IDO or a peptide fragment hereof, wherein said        protein or peptide may be any of the proteins or peptides        described herein    -   iii) determining whether said blood sample comprises antibodies        or T-cells comprising T-cell receptors specifically binding the        protein or peptide    -   iv) thereby determining whether an immune response to said        protein or peptide has been raised in said individual.

The individual is preferably a human being, for example a human beingthat has been immunized with IDO or a peptide fragment hereof or anucleic acid encoding said protein or peptide.

Kit of Parts

The invention also relates to a kit-of-parts comprising

-   -   any of the vaccine compositions described herein and/or    -   an IDO protein or variant hereof and/or    -   any of the polypeptide fragments of IDO, variant hereof, and/or        peptides derived here from as described herein and/or    -   any of the nucleic acids encoding the proteins of the above two        bullet points and instructions on how to use the kit of parts.

The invention also relates to a kit-of-parts comprising

-   -   any of the vaccine compositions described herein and/or    -   an IDO protein or variant hereof and/or    -   any of the polypeptide fragments of IDO, variant hereof, and/or        peptides derived here from as described herein and/or    -   any of the nucleic acids encoding the proteins of the above two        bullet points and a second active ingredient.

Preferably, the second active ingredient is chosen in correspondencewith the clinical condition to be treated so that in the case where acancer is to be treated the second active ingredient is chosen amonge.g. chemotherapeutic agents as listed above. Likewise, if treating amicrobial/viral infection, the second active ingredient is preferably ananti-biotic and/or an anti-viral agent.

The components of the kit-of-parts are preferably comprised inindividual compositions, it is however within the scope of the presentinvention that the components of the kit-of-parts all are comprisedwithin the same composition. The components of the kit-of-parts may thusbe administered simultaneously or sequentially in any order.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1: HLA-A2-restricted T cell responses against IDO as measured byIFN-γ ELISPOT. PBL from 13 healthy individuals, 4 breast cancerpatients, 6 melanoma patients, and 10 renal cell carcinoma patients wereanalyzed. All individuals were HLA-A2 positive. The peptides IDO2(FLVSLLVEI) (SEQ ID NO: 3)(a), IDO6 (VLSKGDGL)(SEQ ID NO: 7) (b), andIDO5 (ALLEIASCL) (SEQ ID NO: 6)(c) were examined. T lymphocytes werestimulated once with peptide before being plated at 4×10⁵ cells per wellin duplicates either without or with the relevant peptide. The averagenumber of peptide-specific spots (after subtraction of spots withoutadded peptide) was calculated for each patient using the ImmunoSpotSeries 2.0 Analyzer (CTL Analyzers) (d) The number of IDO5-specificcells in PBMC measured by IFN-γ ELISPOT in correlation to IDO expressionin the PBMC measured by intracellular IDO stainings. Patients weredivided into two groups hosting; IDO− PBMC or IDO+PBMC. IntracellularIDO expression was given by a onetailed two sampled T-test comparingMFIIDO and MFIIsotype control, where MFI is the Mean FluorescenceIntensity. For p-values<0.05 (significance level) PBMC were definedIDO+. White triangle gives the average number of IDO5-specific spots per4×10⁵ PBMC in each group. Black triangles indicate the average number ofIDO5-specific spots in each group (e) Example of an ELISPOT responseagainst IDO5 in PBMC from a breast cancer patient.

FIG. 2: Tetramer analysis of IDO5-specific T cells. (a), The binding ofthe HLA-A2-restricted positive control peptide HIV-1 pol₄₇₆₋₄₈₄(ILKEPVHGV) was compared with the peptide IDO5 by an assembly assay.(b), An example of IDO5-specific, CD8 T cells in PBL from a renal cellcarcinoma patient visualized by flow cytometry staining using thetetramer complex HLA-A2/IDO5-PE, and CD8-allophycocyanin. As a negativecontrol, PBL from the same patient were stained with the tetramercomplex HLA-A2/HIV pol476-484-PE, and CD8-allophycocyanin. (c), PBL fromhealthy donors or from patients with breast cancer, melanoma cancer orrenal cell carcinoma were stained with the tetramer complex HLA-A2/IDO5or HLA-A2/HIV pol and analyzed by flow cytometry either ex vivo or afterone in vitro peptide stimulation. The dotted lines illustrate that IDO5tetramer positive cells are detectable both ex vivo and in vitro in thesame patients. (d), An example of CD45RA and CD28 phenotype analysis ofIDO5 tetramer/CD8 gated cells from CD8 T cell enriched PBMC from a renalcell carcinoma patient visualised ex vivo by flow cytometry. Forcomparison, the cells were stained with isotype matched controls (e), Anexample of an IL-2 expanded TIL culture from a melanoma patientvisualised by flow cytometry staining using the tetramer complexHLAA2/IDO5-PE, and CD8-APC-Cy7. As a negative control, the TILs werestained with the tetramer complex HLA-A2/HIV pol476-484-PE, andCD8-APC-Cy7. (f), As a positive control of the IDO5 tetramer, anIDO5-specific T-cell clone was stained with the HLA-A2/HIV-PE andHLA-A2/IDO5-PE tetramers.

FIG. 3: Specificity and functional capacity of an IDO5-specific T-cellclone. (RBS35) assayed by ⁵¹Cr-release assay. (a), Lysis of T2-cellswith no peptide or pulsed with IDO5 peptide. (b), Specific lysis of theIDO+, HLA-A2+ colon cancer cell line SW480 without or with the additionof the HLA-class I specific antibody W6/32, and lysis of the IDO−,HLA-A2+ colon cancer cell line HCT-116. (c), Lysis of the IDO+, HLA-A2+melanoma cell line FM55M without and with the addition of cold T2-cellspulsed with IDO5 or unpulsed (inhibitor to target ratio=20:1) (d), Lysisof AML-blasts enriched from an HLA-A2 positive AML patient. AML-blasts,B cells, and T cells were depleted from the bone marrow of the AMLpatient using CD19⁺ and CD3⁺ microbeads, respectively. The highlyenriched AML-blasts were used as target cells with or without theaddition of the HLA-class I specific antibody W6/32. All assays wereperformed in different E:T ratios. (e) Histograms showing intracellularIDO expression in cancer cell lines. Data are representative of 3experiments. Intracellular IDO expression was given by a one-tailed twosampled T-test comparing MFIIDO (dark histograms) and MFIIsotype control(light histograms), where MFI is the Mean Fluorescence Intensity. Left:HCT-116 (p=0.300). Right: SW480 (p=0.002).

FIG. 4: Histograms show intracellular IDO stainings (dark histograms).Negative controls were stainings with the secondary fluorochromeconjugated antibody alone (light histograms). The IDO expression wasdetermined using the staining index defined asMFI_(positive)−MFI_(background)/2× SD_(background) where MFI is meanfluorescence intensity. Cells were defined IDO positive if the stainingindex>1²¹. (a), Colon cancer cell lines HCT-116 (0,01), and SW480 (1,3)(b), breast cancer cell line CAMA-1 (1,3), and CAMA-1+IFN-γ (1,8), and(c), immature DC (0,2), and mature DC (1,2).

FIG. 5: Functional capacity of an IDO5-specific T-cell clone (RBS35) tokill IFN-γ treated breast cancer cell lines assayed by ⁵¹Cr-releaseassay. Lysis of the HLA-A2 positive breast cancer cell lines CAMA-1 (a)and MDA-MB-231 (b) before and after IFN-γ treatment. All assays wereperformed in different E:T ratios. (c), Left: Histograms showingintracellular IDO expression in CAMA-1 before and after IFN-γ treatment.Data are representative of 3 experiments. Intracellular IDO expressionwas given by a one-tailed two sampled T-test comparing MFIIDO (darkhistograms) and MFIIsotype control (light histograms), where MFI is theMean Fluorescence Intensity. Top: CAMA-1 (p=0.020 and MFIIDO/MFIIsotypecontrol=2.3). Bottom: CAMA-1+IFN-γ treatment 25 (p=0.004 andMFIIDO/MFIIsotype control=3.5). Right: Histograms showing HLA-A2expression in CAMA-1 before and after IFN-γ treatment. Data arerepresentative of 3 experiments. HLA-A2 expression was given by aone-tailed two sampled T-test comparing MFIHLA-A2 (dark histograms) andMFIIsotype control (light histograms). Top: CAMA-1 (p=0.004 andMFIHLA-A2/MFIIsotype control=43.7). Bottom: CAMA-1+IFN-γ treatment(p=0.002 and MFIIDO/MFIHLA-A2=141.2). (d), Lysis of the colon cancercell line SW480 transfected with IDO ShRNA for down-regulation of IDOprotein expression by an IDO5-specific T-cell bulk culture. As apositive control, SW480 cells transfected with control ShRNA were usedas target cells. All assays were performed in different E:T ratios. (d),Histograms showing intracellular IDO expression in SW480 transfectedwith control ShRNA (p=0.001 and MFIIDO/MFIIsotype control=4.8) (top) andIDO ShRNA (p=0.040 and MFIIDO/MFIIsotype control=2.1) (bottom).

FIG. 6: Functional capacity of an IDO5-specific T-cell clone (RBS35) tokill DC assayed by ⁵¹Cr-release assay. (a), Lysis of autologous immatureand mature DC. (b), Lysis of HLA-A2+ allogeneic immature and mature DC.All assays were performed in different E:T ratios. (c), Histogramsshowing intracellular IDO expression in DC. Data are representative of 3experiments. Intracellular IDO expression was given by a one-tailed twosampled T-test comparing MFIIDO (dark histograms) and MFIIsotype control(light histograms), where MFI is the Mean Fluorescence Intensity. Left:In vitro immatured DC (p=0.100). Right: In vitro matured DC (p=0.001).(d), Lysis of autologous CD14+ monoctyes, CD3+ T cells and CD19+ B cellsisolated directly ex vivo from IDO+ PBMC. As a control, we used in vitrogenerated autologous IDO− immatured DC and IDO+ matured DC. (e),Examples of HLA-A2 restricted T-cell responses against EBV BMLF1280-288(GLCTLVAML) (SEQ ID NO: 19) as measured by ELISPOT in PBMC from a breastcancer patient. Cultures of PBMC were treated with IFN-γ for 5 dayswithout (left) and with 26 IDO-specific T cells (at a PBMC:IDO-specificT cell ratio of 3000:1) (right) before examination for reactivityagainst the HLA-A2 restricted epitope from EBV BMLF1 (GLCTLVAML) (SEQ IDNO: 19). Three different PMBC concentrations was examined; 1.5×10⁵cells, 5×10⁴ cells (two top rows) and 10⁴ cells (bottom two rows).

FIG. 7: Specificity and functional capacity of IDO5-specific T cellsassayed by 51Cr-release assays: (a), Lysis by RBS35 of the HLA-A2+/IDO+melanoma cell line FM55M without and with the addition of cold T2-cellspulsed with IDO5 peptide or an irrelevant peptide (HIV-1 pol1476-484)(inhibitor to target ratio=20:1), and NK cell activity of RBS35 examinedusing the natural killer cell line K562 as target cells. (b), Lysis byRBS35 of AML-blasts enriched from an HLA-A2+ AML patient. AML-blasts, Bcells, and T cells were depleted from the bone marrow of the AML patientusing CD19+ and CD3+ microbeads, respectively. The highly enrichedAML-blasts were used as target cells with or without the addition of theHLA-class I specific antibody W6/32. (c), Lysis of T2-cells pulsed withIDO5 peptide or an irrelevant peptide (HIV-1 pol1476-484), and lysis ofthe HLAA2+/IDO+ colon cancer cell line SW480 by an IDO5-specific T-cellbulk culture. (d), Lysis of the HLA-A2+/IDO+ colon cancer cell lineSW480 and HLA-A2+/IDO− colon cancer cell line HCT-116 by three differentIDO5-specific T-cell clones (RBS26 (white triangle), RBS31 (blacktriangle), RBS46 (grey triangle)) assayed by 51Cr-release assay. Allassays were performed in different E:T ratios.

FIG. 8: Multiple alignment of IDO sequences by Clustal W

FIG. 9: Pair wise alignment of IDO and IDOLIKE by Clustal W

EXAMPLES Example 1 Patients/Individuals

PBL/PBMC was collected from cancer patients (breast cancer, melanoma,and renal cell carcinoma) and healthy controls. Blood samples were drawna minimum of four weeks after termination of any kind of anti-cancertherapy. The majority of renal cell carcinoma patients had previouslybeen treated with IL2 and IFN-α, most melanoma patients had receivedhigh dose IL2 and IFN-α, while all breast cancer patients werepre-treated with several kinds of chemotherapy, (e.g. epirubicin,docetaxel, cabecitabine), trastuzumab, and/or endocrine therapy. PBLwere isolated using Lymphoprep separation, HLA-typed (Department ofClinical Immunology, University Hospital, Copenhagen, Denmark) andfrozen in FCS with 10% DMSO. A total of 20 HLA-A2+ patients wereincluded, none of these received immunotherapy prior to sampling ofblood. Informed consent was obtained from the patients prior to any oftheses measures.

Peptides

Epitopes from IDO were predicted according to knowledge about preferredpeptide-length, anchor residues and auxiliary anchors of the HLA-A2allele. Scanning of the IDO protein was carried out using the “DatabaseSYFPEITHIP”³² in combination with manual examination of the proteinsequence for MHC class I anchor residues. Selected peptides werepurchased from Genscript. Eleven synthetic 9mer and 10mer peptides wereproduced: IDO1 positions 54-62 (QLRERVEKL (SEQ ID NO: 2)), IDO2positions 164-172 (FLVSLLVEI (SEQ ID NO: 3)), IDO3 positions 195-203(TLLKALLEI (SEQ ID NO: 4)), IDO4 positions 41-49 (FIAKHLPDL (SEQ ID NO:5)), IDO5 positions 199-207 (ALLEIASCL (SEQ ID NO: 6)), IDO6 positions320-328 (VLSKGDGL (SEQ ID NO: 7)), IDO7 positions 383-391 (DLMNFLKTV(SEQ ID NO: 8)), IDO8 positions 275-283 (VLLGIQQTA (SEQ ID NO: 9)), IDO9positions 101-109 (KVLPRNIAV (SEQ ID NO: 10)), IDO10 positions 61-70(KLNMLSIDHL (SEQ ID NO: 11)), and IDO11 positions 341-350 (SLRSYHLQIV(SEQ ID NO: 12)). The peptides were dissolved in DMSO (finalconcentration 10 mM) or distilled water (final concentration 2 mM). TheHLA-A2 high affinity binding epitope HIV-1 pol1476-484 (ILKEPVHGV (SEQID NO: 18)) was used as irrelevant control. The HLA-A2 restrictedEpstein-Barr virus peptide EBVBMLF1280-288 (GLCTLVAML (SEQ ID NO: 19))was used as control.

Assembly Assay for Peptide Binding to MHC Class I Molecules

The binding affinity of the synthetic peptides (Genscript) to HLA-A2molecules, metabolically labelled with [35^(S)]-methionine, was measuredin the assembly assay, as described previously³³. The assay is based onpeptide-mediated stabilization of empty HLA-molecules released upon celllysis, from the TAP-deficient cell line T2. Stably folded HLA-moleculeswere immune-precipitated by using the HLA class-I specific, conformationdependent monoclonal (mAb) W6/32 and separated by isoelectric focusing(IEF) gel electrophoresis. Major histocompatibility complex (MHC)heavy-chain bands were quantified using the ImageGauge Phosphoimagerprogram (FUJI Photo Film, Carrolton, Tex.). The intensity of the band isdirectly related to the amount of peptide-bound class I MHC complexrecovering during the assay. The recovery of HLA-A2 was measured inpresence of 100, 10, 1, and 0.1 μM of the relevant peptide. The C50value was calculated for each peptide as the peptide concentrationsufficient for half maximal stabilization.

Antigen Stimulation of PBL

To extend the sensitivity of the enzyme-linked immunospot (ELISPOT)assay, PBL were stimulated once in vitro with peptide prior toanalysis³⁴. At day 0, PBL were thawed and plated in 2 ml/well at aconcentration of 2×106 cells in 24-well plates (Nunc) in X-vivo medium(BioWhittaker) with 5% heat-inactivated human serum in the presence of10 μM peptide (GenScript). One day later 40 IU/ml recombinantinterleukin-2 (IL-2) (PeproTech) was added to the cultures. The culturedcells were tested in the IFN-γ ELISPOT assay on day 8.

IFN-γ ELISPOT Assay

The ELISPOT assay was used to quantify peptide epitope-specific INF-γreleasing effector cells as described previously¹⁷. In some experimentsPBMC were stimulated once in vitro with peptide prior to analysis asdescribed⁽³⁴⁾ to extend the sensitivity of the assay. Briefly,nitrocellulose bottomed 96-well plates (MultiScreen MAIP N45; Millipore)were coated with anti-IFN-γ Ab (1-D1 K; Mabtech). The wells were washed,blocked by X-vivo medium and the effector cells were added in duplicatesat different cell concentrations, with or without 10 μM peptide. Theplates were incubated overnight. The following day, medium was discardedand the wells were washed prior to addition of biotinylated secondary Ab(7-B6-1-Biotin; Mabtech). The plates were incubated at room temperature(RT) for 2 h, washed, and Avidin-enzyme conjugate (AP-Avidin;Calbiochem/Invitrogen Life Technologies) was added to each well. Plateswere incubated at RT for 1 h and the enzyme substrate NBT/BCIP(Invitrogen Life Technologies) was added to each well and incubated atRT for 5-10 min. Upon the emergence of dark purple spots, the reactionwas terminated by washing with tap water. The spots were counted usingthe ImmunoSpot Series 2.0 Analyzer (CTL Analyzers) and thepeptide-specific CTL frequency could be calculated from the numbers ofspot-forming cells.

Flow Cytometry

For tetramer stainings, PBL from cancer patients and healthy donors aswell as TIL from cancer patients were stimulated once in vitro withpeptide, or analysed directly ex vivo. The CD8 cells were isolated fromPBL using the Dynal CD8 negative isolation kit (Dynal Biotech) at day 7.The resulting T cell cultures were stained with PE coupled tetramer,followed by antibody staining with the flourochrome-coupled mAbs:CD8-allophycocyanin/APC-Cy7, CD3-FITC, CD3-FITC, CD45RO-FITC,CD45RA-PE-Cy5 and CD28-allophycocyanin (BD Immunocytometry Systems).Tetramer stainings were performed in PBS+2% FCS, for 15 min, RT, in thedark, whereas antibody stainings were performed in PBS+2% FCS, 4° C., inthe dark. The MHC tetramer complexes used were: HLA-A2/IDO5 (ALLEIASCL(SEQ ID NO: 20)) and HLA-A2/HIV-1 pol1476-484 (ILKEPVHGV (SEQ ID NO:18)). The samples were analyzed on BD FACS aria, using DIVA software (BDBiosciences).

Cancer cell lines and DC were examined for expression of IDO using flowcytometry. After fixation and permeabilization (Cytofix/Cytoperm, BD),cells were stained with mouse anti-IDO antibody (Millipore Corporation)followed by FITC-labeled anti-mouse secondary antibody (DAKO). For allexperiments, a negative control only stained with the FITC-coupledsecondary antibody was included, to determine the backgroundfluorescence from falsely attached secondary antibodyE andauto-fluorescence. The IDO expression was determined using the stainingindex defined as MFIpositive−MFIbackground/2×SDbackground where MFI ismean fluorescence intensity. Cells were defined IDO positive if thestaining index>1²¹.

Cancer cells were examined for HLA-A2 expression using flow cytometry.Cells were stained with a fluorochrome-coupled HLA-A2 mAb (BDBioscience). For comparison, cells were stained with an isotype matchedcontrol. The samples were analyzed on BD FACS aria, using DIVA software(BD Biosciences). Assuming normality, HLA-A2 expression was given by aone-tailed two sampled T-test comparing MFIHLA-A2 and MFIIsotypecontrol, where MFI is the Mean Fluorescence 9 Intensity. Forp-values<0.05 (significance level) cells were defined HLA-A2+. The foldof expression was defined as MFIHLA-A2/MFIIsotype control.

Dendritic Cells (DC)

DC were generated from PBMC by adherence on culture dishes at 37° C. for60 min in RPMI-1640 enriched with 10% human AB serum. Adherent monocyteswere cultured in RPMI-1640 supplemented with 10% human AB serum in thepresence of IL-4 (1000 U/ml) and GM-CSF (800 U/ml) for 6 days. DC werematured by addition of IL-1β (2 ng/ml), IL-6 (1000 U/ml), TNF-α (10ng/ml), and PGE2 (1 μg/ml).

Establishment of Antigen Specific T-Cell Cultures and Clones

PBL from cancer patients were stimulated with irradiated (25 Gy),IDO5-loaded autologous DC (PBL:DC ratio=3×106: 3×105), with 3 μg/ml β2m,20 U/ml IL-12 (PeproTech), and 40 U/ml IL-7 (PeproTech). The culturesgot stimulated every 10 days with irradiated autologous DC (2×) followedby irradiated PBL (2×). 20 U/ml IL-12 (PeproTech) and 40 U/ml IL-7(PeproTech) was added after each stimulation with DC, and 40 U/ml IL-2(PeproTech) was added after each stimulation with PBL. After one monthgrowing cultures were tested for specificity for IDO5 in a standard51Cr-release assay. PBL from a specific culture were cloned by limitingdilution in the presence of 106/ml irradiated (25 Gy) IDO5 loaded PBL,and 120 U/ml IL-2 (PeproTech). Every 3-4 days 50 μl fresh media wereadded containing IL-2 to a final concentration of 120 U/ml. Growingclones were expanded using IDO5 loaded PBL (5×10⁴ cells/well) and 120U/ml IL-2. After expansion the clones were tested for specificity andcytotoxic potential in a standard ⁵¹Cr-release assay.

Cytotoxicity Assay

Conventional ⁵¹Cr-release assays for CTL-mediated cytotoxicity wascarried out as described elsewhere 35. Target cells were T2-cells, invitro generated autologous immature and mature DC, allogeneic HLA-A2positive immature and mature DC, autologous ex vivo isolated monocytes,T cells and B cells (isolated using CD14+, CD3+ or CD19+ microbeads(MACS)), the natural killer target cell line K562, ex vivo enrichedHLA-A2 positive AML-blasts (isolated from the bone marrow of the AMLpatient using CD19+ and CD3+ microbeads (MACS)), the HLA-A2 positivebreast cancer cell lines CAMA-1 and MDA-MB-231, the HLA-A2 positivecolon cancer cell lines HCT-116 and SW480 (all available at the AmericanType Culture Collection (ATCC)), and the HLA-A2 positive melanoma cellline FM55M (from the IPD-ESTDAB database, available atwww.ebi.ac.uk/cgi-bin/ipd/estdab/³⁶). Lysis were blocked using the HLAspecific mAb W6/32 (2 μg/100 μl)³⁷. In some assays, cancer cells weretreated with 100 U/ml IFN-γ for 2 days.

Enrichment of AML Blasts

We depleted B and T cells from the bone marrow of the AML patient usingCD19+ and CD3+ microbeads (MACS), respectively. The highly enrichedAML-blasts (CD3−, CD19−) were used as target cells in a standard ⁵¹Crrelease assay.

Down-Regulation of IDO in Cancer Cells

Human SW480 were transfected with indicated short hairpin RNA (ShRNA)plasmids obtained from SuperArray using FuGene6 (Roche) according tomanufacturers instructions. Cells were lysed directly in LSB buffer(Sigma). The LSB lysates were boiled for 5 min. and loaded on 10%precast protein gels (BioRad). Proteins were electro transferred to aPVDF membrane (Millipore Corporation) by a semidry transfer method andprobed with indicated antibodies according to manufacturersinstructions. Blots were developed with the ECL system obtained fromAmersham and a CCD camera (LAS-1000, Fujifilm). Following antibodieswere used: anti-Cdk7 (MO-1) (Santa Cruz) and anti-IDO (MilliporeCorporation).

IDO-Derived HLA-A2-Restricted T-Cell Epitopes

Eleven IDO-derived peptides were selected using algorithms based on themain HLA-A2 specific anchor residues and subsequently synthesized¹⁶.Using the ELISPOT IFN-γ secretion assay, we then examined peripheralblood T cells from cancer patients and healthy individuals for thepresence of specific T-cell responses against these IDO-derivedpeptides. This approach has previously proved to be highly effective foridentifying tumor specific cytotoxic T-lymphocytes (CTL) in cancerpatients¹⁷⁻¹⁹. Thus, peripheral blood lymphocytes (PBL) from HLA-A2positive, late stage cancer patients (breast cancer, melanoma and renalcell carcinoma) were stimulated once with the different peptides invitro before examination by ELISPOT. This procedure was chosen to extendthe sensitivity of the ELISPOT as described^(17,20). ELISPOT responseswere detected against IDO2 (IDO164-172; FLVSLLVEI (SEQ ID NO: 3)), IDO6(IDO 320-328; VLSKGDGL (SEQ ID NO: 7)), and especially IDO5 (IDO199-207;ALLEIASCL (SEQ ID NO: 6)) (FIG. 1). As control, we examined PBL fromhealthy individuals for reactivity against these three IDO derivedpeptides. No spontaneous responses could be detected against any of theIDO derived peptides in any of the healthy controls. A BLAST search ofthe amino acid sequences of these peptides using the “NCBI database”showed that these motifs are only prevalent in the IDO protein.

Example 2 Materials and Methods are as Described in Example 1 Detectionof IDO-Reactive HLA-A2-Restricted T Cells in Cancer Patients

The apparently most immunogenic IDO-derived peptide, i.e. IDO5, wasexamined for its binding affinity to HLA-A2 by comparison with a HLA-A2high affinity positive control epitope, i.e. HIV-1 pol476-484 (ILKEPVHGV(SEQ ID NO: 18), by the assembly assay (FIG. 2a ). Notably, IDO5 boundHLA-A2 even better than the high-affinity control epitope. The highbinding affinity of IDO5 to HLA-A2 enabled us to make stable HLA-A2/IDO5tetramers, which were used to detect IDO-reactive CTL by flow cytometry.This analysis clearly confirmed the presence of IDO5-reactive CD8 Tcells in the blood of HLA-A2 positive cancer patients (FIG. 2). FIG. 2billustrates an example of an IDO5 specific T cell response after invitro stimulation in a renal cell carcinoma patient with an HIVtetramer-complex used as control. While the frequency of IDO-reactive Tcells are markedly increased by in vitro stimulation, IDO-reactive Tcells were readily detectable ex vivo in selected patients (FIG. 2c ):In the three patients with strongest responses after in vitrostimulation, a respective reactivity was also detected ex vivo. Overall,PBL from 7 HLA-A2 positive healthy individuals and 11 HLA-A2 positivepatients were analyzed which revealed an average frequency of 0.03% IDOreactive cells of total CD8+ T cells after in vitro stimulation incancer patients, compared to 0.001% in healthy donors (FIG. 2c ).

No IDO-reactive T cells could be detected in any of the healthy donors(FIG. 2b ). The ex vivo stainings of IDO-reactive T cells showed thatnaturally occurring IDO5-specific T cells have a CD45RA-CD28+central/effector memory phenotype⁽³⁴⁾. An example of such an ex vivophenotype staining of IDO5 tetramer gated cells is shown in FIG. 2 d.

As a comparison the sample were stained with isotype matched controls.Next, we examined the presence of IDO5− specific T cells in IL-2 treatedTIL cultures from HLA-A2+ melanoma and head and neck cancer patients bytetramer stainings. As illustrated in FIG. 2e IDO5-specific T cellscould readily be detected among the TIL. Overall, 4 of the 5 analyzedpatients had detectable IDO5-specific T cells. Likewise, IDO5-specific Tcells in TIL cultures from melanoma and head and neck cancer patientscould be detected in ELISPOT (data not shown). To control thespecificity of the HLA-A2/IDO5 tetramer we stained an IDO5-specificT-cell clone. The HLA-A2/IDO5 tetramer did efficiently stain theIDO5-specific T-cell clone, whereas the T-cell clone was not stained bythe control HLAA2/HIV tetramer (FIG. 2f ).

Example 3 Materials and Methods are as Described in Example 1 FunctionalCapacity of IDO Specific T-Cells

Having identified patients hosting responses against the IDO5 peptide,we used PBL from such patients to generate CTL bulk cultures againstthis peptide in vitro. PBL were stimulated by autologous IDO5-pulsed DC.After four rounds of stimulation, the peptide specificity was tested instandard 51Cr release assays. Cells from these bulk cultures lysedTAP-deficient T2-cells pulsed with IDO5 peptides. To analyze the lyticcapacity of IDO specific T-cells in more detail, CTL clones wereestablished from these bulk cultures by limiting dilution cloning. Aftera short expansion period, the specificity of the growing clones wasanalyzed in standard 51Cr release assays. Of thirty three T-cell clonesdisplaying an IDO specific lytic capacity, four clones were selected forfurther expansion due to a superior growth rate. A representative T-cellclone is depicted in FIG. 3a : the T-cell clone RBS35 effectively killedIDO5-pulsed T2-cells whereas T2-cells without peptide were not lysed(FIG. 3a ).

Example 4 Materials and Methods are as Described in Example 1 Killing ofTumor Targets by IDO-Specific T Cells

A number of cancer cell lines and DC were examined for IDO expression byintracellular protein staining followed by FACS analysis²¹. To this end,the colon cancer cell line SW480, the melanoma cell line FM55M, thebreast cancer cell lines CAMA-1 and MDA-MB231, directly enrichedAML-blasts, and mature DC were IDO positive. Only the colon cancer cellline HCT-116 and immature DC were IDO negative. Furthermore, IFN-γtreatment of the cancer cell lines increased the IDO expression.Representative examples of IDO stainings are illustrated in histogramsin FIG. 4.

Importantly, the T cell clone RBS35 killed not only peptide pulsedT2-cells but also the HLA-A2+, IDO+ colon cancer cell line SW480 (FIG.3b ) with high efficacy. In contrast, RBS35 did not lyse theHLA-A2+/IDO− colon cancer cell line HCT-116 (FIG. 3b ). HLA-restrictionof RBS35 was confirmed by blocking HLA-class I using the HLA specificmAb W6/32, which completely abolished lysis of the SW480 target cells(FIG. 3b ). Similarly, the HLA-A2+/IDO+ melanoma cell line FM55M waskilled by RBS35 (FIG. 3c ). Cold targeted inhibition assays usingunlabeled T2-cells pulsed with the IDO5 (10 μM) peptide confirmed theHLA-A2/peptide-specificity of the killing: The addition of cold(unlabeled) IDO5-pulsed T2-cells completely abrogated the killing ofFM55M melanoma cells, whereas the addition of cold T2-cells withoutpeptide did not have an effect on the killing of FM55M (FIG. 3c ).Neither did the addition of cold T2-cells pulsed with an irrelevantpeptide (HIV-1 pol1476-484) did not have an effect on the killing ofFM55M (FIG. 7a ). No cytotoxicity was observed against the NK-celltarget cell line K562 (FIG. 7a ).

Furthermore, we tested the ability of RBS35 to lyse human HLA-A2+AML-blasts enriched directly ex vivo from the bone-marrow of AMLpatients. For this purpose, we depleted T cells (CD3+) and B cells(CD19+) from the bone marrow of HLA-A2+ AML patients; the highlyenriched AML-blasts (CD3−, CD19−) were subsequently used as target cellsin a ⁵¹Cr release assay. As shown in FIG. 3d ; RBS35 efficiently lysedthe leukemia cells in an HLA-dependent manner. We enriched AML blastsfrom six patients (5 HLA-A2+ patients and 1 HLA-A2− patient) and allthese expressed IDO (data not shown). RBS35 efficiently lysed theHLA-A2+ leukemia cells in an HLA-dependent manner, while HLA-A2−leukemia cells were not lysed (FIG. 7b ).

To illustrate the representative killing of tumor targets by RBS35 thekilling of SW480 by a polyclonal, IDO5-specific bulk culture as well asthree other T-cell clones (RBS26, RBS31, RBS46) are shown in FIG. 7c andFIG. 7d . Similar to RBS35, none of these clones (RBS26, RBS31, RBS46)lysed the HLA-A2+/IDO− colon cancer cell line HCT-116 (FIG. 7d ).

Finally, we examined the killing of the HLA-A2+ breast cancer cell linesCAMA-1 and MDA-MB-231. The CAMA-1 cell line was killed by RBS35 (FIG. 5a), whereas MDA-MB-231 was not recognized by RBS35 (FIG. 5b ). INF-γtreatment increased the expression of IDO in both cell lines. Inagreement with this, INF-γ treatment increased the killing by RBS35 ofCAMA-1 and introduced killing of the MDA-MB-231 cells (FIG. 5).

Additionally, we show that killing by a polyclonal IDO5-specific bulkculture and the RBS35 clone are indeed IDO-specific. Thus, using IDOshRNA we down-regulated IDO protein expression in the human SW480 coloncancer cell line and thereby rescue these tumor cells from being killed(FIG. 5d ). This down-regulation was visualized by intracellular proteinstainings. These stainings confirmed that the use of IDO ShRNA reducedthe level of IDO protein expression in the cells (FIG. 5e ).Subsequently, the transfected cells were used as target cells in a51Cr-release assay. Cancer cells transfected with IDO ShRNA were notrecognized by the polyclonal IDO-specific bulk culture, whereas cellstransfected with irrelevant control ShRNA were killed as illustrated inFIG. 5 d.

Example 5 Materials and Methods are as Described in Example 1 Killing ofImmune Competent Cells by IDO-Specific T Cells

IDO expression is not restricted to tumor and tumor stroma cells, butcan also be induced in immune cells. Thus, as the next and even moreimportant step we addressed the question whether IDO-expressing DC wouldalso be susceptible killing by IDO-reactive CTL. To test this notion, wegenerated autologous DC from the same donors from whom the CTL cloneshad been generated; the DC were matured by the addition of a standardmaturation cocktail consisting of IL-113, IL-6, TNF-α, and PGE2²². RBS35effectively killed the matured DC. In contrast, autologous immature IDO−DC were not killed by RBS35 (FIG. 6a ). Moreover, we examined therecognition of IDO+ mature DC as well as IDO− immature DC from anHLA-A2+ donor by RBS35. The allogenic matured DC were killed by RBS35whereas the IDO− immature DC from the same donor (FIG. 6b ).

In FIG. 6c it is illustrated that mDC express IDO in contrast to iDC.Next, we tested the ability of RBS35 to lyse autologous monoctyes, Tcells and B cells. For this purpose, we isolated CD14+ monoctyes, CD3+ Tcells and CD19+ B cells directly ex vivo from IDO+ PBMC. The isolatedcells were subsequently used as target cells in a 51Cr-release assay.Autologous CD14+ monoctyes, CD3+ T cells and CD19+ B cells were notlysed by RBS35 (FIG. 6d ).

Finally, we sat up an in vitro model to examine if IDO-specific T cellsenhance immune responses by depleting IDO-expressing suppressive cells.Hence, cultures of PBMC were treated with IFN-γ to increase the immuneactivity as well as IDO expression in the cultures with and withoutautologous IDO specific T cells. Five days later we examined the immunereactivity against the HLA-A2 restricted immunodominant epitope from EBVBMLF1280-288 (GLCTLVAML)(SEQ ID NO: 19) in the cultures. Although theoverall cell number was the same in the cultures the reactivity againstthe EBV peptide was higher in the cultures with IDO-specific T cells(FIG. 6e ). Next, we scrutinized if the addition of IDO specific T cellsincreased the immune reactivity to an extent that allowed detection ofEBV responses in an ELISPOT with only 104 PBMC far below the normaldetection limit. Indeed, we could detect a clear EBV response even atthis low concentration of PBMC (FIG. 6e ). As expected we could notdetect any EBV response at this low cell concentration in the culturewithout IDO-specific T cells (FIG. 6e ).

REFERENCE LIST

-   Popov & Schultze; J Mol Med. 2008 February; 86(2):145-60. Epub 2007    September 18-   Nicolette C A, Healey D, Tcherepanova I, Whelton P, Monesmith T,    Coombs L, Finke L H, Whiteside T, Miesowicz F, (2007). Dendritic    cells for active immunotherapy: optimizing design and manufacture in    order to develop commercially and clinically viable products.    Vaccine, September 27; 25 Suppl 2:B47-60. Epub 2007-   Walter E A, Greenberg P D, Gilbert M J, Finch R J, Watanabe K S,    Thomas E D, Riddell S R (1995). Reconstitution of cellular immunity    against cytomegalovirus in recipients of allogeneic bone marrow by    transfer of T-cell clones from the donor. N Engl J Med. 1995 Oct.    19; 333(16):1038-44-   Morgan R A, Dudley M E, Wunderlich J R, Hughes M S, Yang J C, Sherry    R M, Royal R E, Topalian S L, Kammula U S, Restifo N P, Zheng Z,    Nahvi A, de Vries C R, Rogers-Freezer L J, Mavroukakis S A,    Rosenberg S A. Cancer regression in patients after transfer of    genetically engineered lymphocytes. Science. 2006 Oct. 6;    314(5796):126-9. Epub 2006 Aug. 31.-   Schaft N, Dörrie J, Müller I, Beck V, Baumann S, Schunder T, Kämpgen    E, Schuler G. A new way to generate cytolytic tumor-specific T    cells: electroporation of RNA coding for a T cell receptor into T    lymphocytes. Cancer Immunol Immunother. 2006 September;    55(9):1132-41. Epub 2005 Dec. 13-   1. Morris E, Hart D, Gao L, et al: Generation of tumor-specific    T-cell therapies. Blood Rev 20:61-69, 2006-   3. Platten M, Ho P P, Youssef S, et al: Treatment of autoimmune    neuroinflammation with a synthetic tryptophan metabolite. Science    310:850-855, 2005-   4. Bauer T M, Jiga L P, Chuang J J, et al: Studying the    immunosuppressive role of indoleamine 2,3-dioxygenase: tryptophan    metabolites suppress rat allogeneic T-cell responses in vitro and in    vivo. Transpl Int 18:95-100, 2005-   7. Sharma M D, Baban B, Chandler P, et al: Plasmacytoid dendritic    cells from mouse tumor-draining lymph nodes directly activate mature    Tregs via indoleamine 2,3-dioxygenase. J Clin Invest 117:2570-2582,    2007-   8. Munn D H, Sharma M D, Hou D, et al: Expression of indoleamine    2,3-dioxygenase by plasmacytoid dendritic cells in tumor-draining    lymph nodes. J Clin Invest 114:280-290, 2004-   9. Thebault P, Condamine T, Heslan M, et al: Role of IFNgamma in    allograft tolerance mediated by CD4+CD25+ regulatory T cells by    induction of IDO in endothelial cells. Am J Transplant 7:2472-2482,    2007-   10. Baban B, Hansen A M, Chandler P R, et al: A minor population of    splenic dendritic cells expressing CD19 mediates IDO-dependent T    cell suppression via type I IFN signaling following B7 ligation. Int    Immunol 17:909-919, 2005-   11. Zou W: Immunosuppressive networks in the tumour environment and    their therapeutic relevance. Nat Rev Cancer 5:263-274, 2005-   12. Uyttenhove C, Pilotte L, Theate I, et al: Evidence for a tumoral    immune resistance mechanism based on tryptophan degradation by    indoleamine 2,3-dioxygenase. Nat Med 9:1269-1274, 2003-   13. Okamoto A, Nikaido T, Ochiai K, et al: Indoleamine    2,3-dioxygenase serves as a marker of poor prognosis in gene    expression profiles of serous ovarian cancer cells. Clin Cancer Res    11:6030-6039, 2005-   14. Weinlich G, Murr C, Richardsen L, et al: Decreased serum    tryptophan concentration predicts poor prognosis in malignant    melanoma patients. Dermatology 214:8-14, 2007-   15. Lob S, Konigsrainer A, Schafer R, et al: Levo- but not    dextro-1-methyl tryptophan abrogates the IDO activity of human    dendritic cells. Blood: 2007-   16. Andersen M H, Tan L, Sondergaard I, et al: Poor correspondence    between predicted and experimental binding of peptides to class I    MHC molecules. Tissue Antigens 55:519-531, 2000-   17. Andersen M H, Pedersen L O, Becker J C, et al: Identification of    a Cytotoxic T Lymphocyte Response to the Apoptose Inhibitor Protein    Survivin in Cancer Patients. Cancer Res 61:869-872, 2001-   18. Scheibenbogen C, Sun Y, Keilholz U, et al: Identification of    known and novel immunogenic T-cell epitopes from tumor antigens    recognized by peripheral blood T cells from patients responding to    IL-2-based treatment. Int J Cancer 20; 98:409-414, 2002-   19. Herr W, Ranieri E, Gambotto A, et al: Identification of    naturally processed and HLA-presented Epstein-Barr virus peptides    recognized by CD4(+) or CD8(+) T lymphocytes from human blood. Proc    Natl Acad Sci USA 96:12033-12038, 1999-   20. Keilholz U, Weber J, Finke J H, et al: Immunologic monitoring of    cancer vaccine therapy: results of a workshop sponsored by the    Society for Biological Therapy. J Immunother 25:97-138, 2002-   21. Maecker H T, Frey T, Nomura L E, et al: Selecting fluorochrome    conjugates for maximum sensitivity. Cytometry A 62:169-173, 2004-   22. Nguyen X D, Eichler H, Sucker A, et al: Collection of autologous    monocytes for dendritic cell vaccination therapy in metastatic    melanoma patients. Transfusion 42:428-432, 2002-   23. Hwang S L, Chung N P, Chan J K, et al: Indoleamine 2,    3-dioxygenase (IDO) is essential for dendritic cell activation and    chemotactic responsiveness to chemokines.

Cell Res 15:167-175, 2005

-   24. Boasso A, Herbeuval J P, Hardy A W, et al: HIV inhibits CD4+    T-cell proliferation by inducing indoleamine 2,3-dioxygenase in    plasmacytoid dendritic cells. Blood 109:3351-3359, 2007-   25. Wobser M, Voigt H, Houben R, et al: Dendritic cell based    antitumor vaccination: impact of functional indoleamine    2,3-dioxygenase expression. Cancer Immunol Immunother 56:1017-1024,    2007-   26. Popov A, Schultze J L: IDO-expressing regulatory dendritic cells    in cancer and chronic infection. J Mol Med.: 2007-   27. Scheler M, Wenzel J, Tuting T, et al: Indoleamine    2,3-dioxygenase (IDO): the antagonist of type I interferon-driven    skin inflammation? Am J Pathol 171:1936-1943, 2007-   28. Choi B K, Kim Y H, Kang W J, et al: Mechanisms involved in    synergistic anticancer immunity of anti-4-1BB and anti-CD4 therapy.    Cancer Res 67:8891-8899, 2007-   29. Beck K E, Blansfield J A, Tran K Q, et al: Enterocolitis in    patients with cancer after antibody blockade of cytotoxic    T-lymphocyte-associated antigen 4. J Clin Oncol %20; 24:2283-2289,    2006-   30. Sanderson K, Scotland R, Lee P, et al: Autoimmunity in a phase I    trial of a fully human anti-cytotoxic T-lymphocyte antigen-4    monoclonal antibody with multiple melanoma peptides and Montanide    ISA 51 for patients with resected stages III and IV melanoma. J Clin    Oncol 23:741-750, 2005-   31. Maker A V, Phan G Q, Attia P, et al: Tumor regression and    autoimmunity in patients treated with cytotoxic T    lymphocyte-associated antigen 4 blockade and interleukin 2: a phase    I/II study. Ann Surg Oncol 12:1005-1016, 2005-   32. Rammensee H G, Falk K, Roetzschke O: MHC molecules as peptide    receptors. Curr Biol 5:35-44, 1995-   33. Elvin J, Cerundolo V, Elliott T, et al: A quantitative assay of    peptide-dependent class I assembly. Eur J Immunol 21:2025-2031, 1991-   34. McCutcheon M, Wehner N, Wensky A, et al: A sensitive ELISPOT    assay to detect low-frequency human T lymphocytes. J Immunol Methods    210:149-166, 1997-   35. Andersen M H, Bonfill J E, Neisig A, et al: Phosphorylated    Peptides Can Be Transported by TAP Molecules, Presented by Class I    MHC Molecules, and Recognized by Phosphopeptide-Specific CTL. J    Immunol 163:3812-3818, 1999-   36. Pawelec G, Marsh S G: ESTDAB: a collection of immunologically    characterised melanoma cell lines and searchable databank. Cancer    Immunol Immunother 55:623-627, 2006-   37. Schmidt S M, Schag K, Muller M R, et al: Survivin is a shared    tumor-associated antigen expressed in a broad variety of    malignancies and recognized by specific cytotoxic T cells. Blood    102:571-576, 2003

1. A vaccine composition, comprising: a) an isolated immunogenicallyactive peptide fragment of Indoleamine 2,3-dioxygenase (IDO) of SEQ IDNO: 1, wherein the amino acid sequence of the peptide fragment consistsof at the most 30 consecutive amino acid residues of SEQ ID NO: 1, or anucleic acid encoding said peptide fragment; and a) an adjuvant for useas a medicament.
 2. The vaccine composition according to claim 1,wherein the amino acid sequence of the isolated immunogenically activepeptide fragment comprises amino acid residues 199-207 of SEQ ID NO: 1.3. (canceled)
 4. The vaccine composition according to claim 2, whereinsaid immunogenically active peptide fragment is a) a consecutivesequence in the range of 8 to 11 amino acids and/or b) a consecutivesequence in the range of 18 to 30 amino acids of said IDO of SEQ IDNO:1.
 5. The vaccine composition of according to claim 2, wherein thevaccine composition is capable of eliciting an immune response against acancer and/or an antigen presenting cell expressing IDO of SEQ ID NO: 1when administered to an individual suffering from a clinical conditioncharacterized by expression of IDO.
 6. (canceled)
 7. The vaccineaccording to claim 5, wherein the clinical condition is selected fromcancer or an infection.
 8. (canceled)
 9. The vaccine compositionaccording to claim 2, wherein the peptide fragment is a majorhistocompatibility complex (MHC) Class I-restricted peptide or an MHCClass II-restricted peptide having at least one of the followingcharacteristics: a) capable of eliciting I gamma-interferon(INF-γ)-producing cells in a peripheral blood lymphocyte (PBL)population of an individual suffering from a clinical condition at afrequency of at least 1 per 10⁴ PBLs as determined by an enzyme-linkedimmunospot (ELISPOT) assay, and/or b) capable of in situ detection in atumor tissue of cytotoxic T-lymphocytes (CTLs) that are reactive withthe epitope peptide, and/or c) capable of inducing the growth ofIDO-specific T-cells in vitro.
 10. The vaccine composition according toclaim 2, wherein the peptide fragment is restricted by a majorhistocompatibility complex (MHC) Class II molecule.
 11. (canceled) 12.The vaccine composition according to claim 3, comprising a peptidefragment, wherein the peptide fragment consists of 8 to 10 or 18 to 25consecutive amino acids from IDO of SEQ ID NO:1.
 13. (canceled)
 14. Thevaccine composition according to claim 2, wherein the peptide fragmentis capable of eliciting INF-γ-producing cells in a PBL population of anindividual suffering from a clinical condition where IDO of SEQ ID NO: 1is expressed.
 15. The vaccine composition according to claim 6, whereinthe cancer is a tumor forming cancer. 16-19. (canceled)
 20. The vaccinecomposition according to claim 2, where the vaccine elicits theproduction in a vaccinated individual of regulatory T-cells having acytotoxic effect against the IDO expressing cancer cells and/or IDOexpressing antigen presenting cells.
 21. (canceled)
 22. The vaccinecomposition according to claim 2, wherein the vaccine composition iscapable of eliciting a clinical response in a subject, wherein theclinical response is comprised by a stable disease, a partial responseor complete remission. 23-24. (canceled)
 25. The vaccine compositionaccording to claim 2, wherein the adjuvant is selected from the groupconsisting of a Montanide ISA adjuvant and GM-CSF.
 26. The vaccinecomposition according to claim 25, wherein the Montanide ISA adjuvant isselected from Montanide ISA 51 or Montanide ISA
 720. 27-28. (canceled)29. The vaccine composition according to claim 2, wherein the vaccinecomposition comprises antigen presenting cells loaded with theimmunogenically active peptide fragment, or the nucleic acid encodingsaid immunogenically active peptide fragment. 30-34. (canceled)
 35. Akit-of-parts comprising the vaccine composition according to claim 1,and a second active ingredient. 36-38. (canceled)
 39. The kit-of-partsaccording to claim 35, wherein the second active ingredient is ananti-cancer agent. 40-41. (canceled)
 42. The kit-of-parts according toclaim 35, wherein the second active ingredient is an antibiotic. 43-73.(canceled)
 74. An isolated immunogenically active peptide fragment ofIndoleamine 2,3-dioxygenase (IDO) of SEQ ID NO: 1, wherein the aminoacid sequence of the fragment consists of up to 30 consecutive aminoacid residues of SEQ ID NO:
 1. 75. The isolated immunogenically activepeptide fragment of claim 74, wherein the consecutive amino acidsequence consists of said immunogenically active peptide fragment is a)a consecutive sequence in the range of 8 to 11 amino acids and/or b) aconsecutive sequence in the range of 18 to 30 amino acids of said IDO ofSEQ ID NO:
 1. 76. The active peptide fragment of claim 74, wherein theconsecutive amino acid residues comprise amino acid residues 199-207 ofSEQ ID NO: 1.